Insulin-like growth factor 1 receptor -specific antibodies and uses thereof

ABSTRACT

The blood-brain barrier (BBB) prevents transport of molecules larger than 500 Daltons from blood to brain. Receptor-mediated transcytosis (RMT) facilitates transport across the BBB of specific molecules that bind receptors on brain endothelial cells that form the BBB. An insulin-like growth factor 1 receptor (IGF1R)-binding antibody or fragment thereof is identified that transmigrates the BBB by RMT. The antibody or fragment is used to deliver a cargo molecule across the BBB, wherein the cargo molecule may be a therapeutic or detectable agent. The antibody is a camelid VHH, prepared by immunizing a llama with a 933-amino acid IGF1R polypeptide. Humanized forms of the camelid VHH are also generated.

FIELD OF THE INVENTION

The present invention relates to Insulin-Like Growth Factor 1Receptor-specific antibodies, fragments thereof, and uses thereof. Morespecifically, the present invention relates to Insulin-Like GrowthFactor 1 Receptor-specific antibodies and fragments thereof thattransmigrate the blood-brain barrier, and uses thereof.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases, such as Alzheimer's and Parkinson's disease,are an increasing burden on our ageing society because there arecurrently no effective treatments for these disabling conditions.Treatment as well as early diagnosis of these and other diseases thatoriginate in the brain remain challenging because the majority ofsuitable therapeutic molecules and diagnostics cannot penetrate thetight and highly restrictive blood-brain barrier (BBB) (Abbott, 2013).The BBB constitutes a physical barricade that is formed by brainendothelial cells (BECs) that line the blood vessels and connect witheach other through tight junctions (Abbott, 2013). The tight junctionsformed between the BECs are essential for the integrity of the BBB andprevent the paracellular transport of molecules larger than 500 daltons(Da). Because brain endothelial cells exhibit very low pinocytosis rates(Abbott, 2013), transcellular transport of larger molecules is limitedto the highly specific receptor mediated transcytosis (RMT) pathway, andthe passive, charge-based adsorption mediated transcytosis (Abbott,2013; Pardridge, 2002). Additionally, the high density of efflux pumps,such as P-glycoprotein or the multi-drug resistance protein-1 (MDR-1),contribute to the removal of unwanted substances from the brain (Abbott,2013).

While all these characteristics protect the brain from pathogens andtoxins, they equally prevent the entry of most therapeutics. In fact,less than 5% of small molecule therapeutics and virtually none of thelarger therapeutics can cross the BBB in pharmacologically relevantconcentrations (i.e., sufficient to engage a central nervous system(CNS) target and elicit pharmacologic/therapeutic response) unless theyare specifically ‘ferried’, that is, coupled to a transporter molecule.Due to the lack of effective ‘carriers’ to transport molecules acrossthe BBB, numerous drugs against neurodegenerative diseases have been‘shelved’ or eliminated from further development as they cannot bedelivered to the brain in sufficient amount.

Different approaches to deliver larger molecules into the brain havebeen explored. For example, the integrity of the BBB may be disrupted,resulting in a leaky BBB, which in turn allows for unrestricted,paracellular entry of larger molecules into the brain. Tight junctionscan be successfully loosened or disrupted by various approaches. Forexample, injection of substances that induce osmotic shock (for example,mannitol, hypertonic solutions) into the blood stream causes cellshrinkage and results in the disruption of tight junctions, thereforeseverely compromising the BBB (Guillaume, 2010). Other modulators oftight junctions include alkylglycerols, bradykinin and several analoguesthereof, as well as viruses that modulate expression of proteinsinvolved in maintaining the tight junctions (Erdlenbruch et al., 2003;Preston et al., 2008; Gan et al., 2013). A more localized disruption ofthe BBB is possible through application of ultrasound (Nhan et al.,2013). However, the periods during which the BBB is disrupted aresufficient to alter brain homeostasis and allow harmful chemicals,toxins and pathogens to enter the brain; this can result in seriousside-effects, e.g., seizures and brain swelling, infection and possiblypermanent neuropathological changes. As would be evident to those ofskill in the art, repeated treatments with these techniques for chronicand diffuse brain diseases affecting multiple brain regions are notpractical. Most of these treatments are costly, necessitatehospitalisation, and some approaches require anesthesia.

Another approach for circumventing the BBB is direct injection oftherapeutic molecules into the cerebrospinal fluid (CSF), theparenchymal space, or other parts of the brain. Several delivery methodshave been developed, including: intracerebral (intra-parenchymal),intraventricular, and intrathecal delivery via infusion orconvection-enhanced diffusion (CED) pumps. However, any type of directinjection into the brain or intracerebral implant is an invasive andcostly procedure, as it requires hospitalization, anesthesia, and oftensurgery. Moreover, the poor diffusion rates of the therapeutics,particularly large biologics, within brain parenchyma limit thepenetration of therapeutics to only small areas surrounding the site ofinjection/implantation. The correct placement of injections, catheters,and implants is challenging yet crucial to achieve diffusion of the drugto the targeted region of the brain. Additionally, catheters andimplants provide a site for infection and/or immune response against theforeign material.

In another attempt to increase delivery across the BBB, CNS drugs havebeen modified to increase their brain uptake. Such modifications caninclude a change of their surface charge, a reduction in molecule size,and change to the lipohilicity of the drugs. However, any modificationsto increase brain penetration are also likely to alter the overallpharmacology of the drug, including its desired activity and/orspecificity. In addition, lipophilic molecules are more prone to beingexported from the brain through the P-glycoprotein efflux pump.

Finally, endogenous transport mechanisms across the BBB have beenexploited. Physiological mechanisms that allow transport of largemolecules across the BBB can be divided into the highly specificreceptor mediated transcytosis (RMT) and the non-specific charge basedadsorptive mediated endocytosis pathways. Endocytosis is triggered uponbinding of the specific ligand to its receptor, or upon electrostaticinteraction between the cationic ligand or drug and the anionicfunctional groups on the brain endothelial cell surface (luminal side),respectively. Subsequently, the newly formed endosome is transcytosedacross the cell to the abluminal side, to release its cargo.

Because adsorptive mediated transcytosis is non-specific,charge-mediated interaction, it occurs in all vascular beds and organs,limiting the availability of drug for brain delivery. Therefore,exploiting the RMT pathway remains the only physiological, non-invasiveyet highly receptor-specific brain delivery method.

Only a few receptors are presently known to undergo RMT at the BBB and‘ferry’ across their natural ligands: the well-studied transferrinreceptor (TfR), the insulin receptor (IR), low-density lipoproteinreceptor related proteins 1 and 2 (LRP-1 and -2), diphtheria toxinreceptor, and TMEM30A. Peptides, natural ligands, and antibodies orantibody fragments have been developed that bind to these receptors(Pardridge et al., 1991; Yu et al., 2011; Muruganandam et al., 2001;Abulrob et al., 2005; Demeule, 2008; Sumbria et al., 2013), functioningas drug-to-brain transporters that utilize endogenous RMT pathways.However, to date only a single peptide (Angiopep ANG1005, targetingLRP-1) has been analyzed in phase I clinical studies, while othercandidates are being studied in laboratory settings. The RMT pathwayappears to be the most promising pathway for drug transport to thebrain, but current approaches have limitations, including: non-selectiveexpression of the target receptor at the BBB, competition between thecarrier and the natural ligands to the receptor, ineffectivetranscytosis of a receptor as well as lysosomal degradation ofendocytosed carriers (Xiao and Gun, 2013).

The lack of high-capacity and high-selectivity BBB carriers delays thedevelopment of new therapeutics and diagnostics for diseases originatingin the brain, including brain tumors and neurodegenerative diseases.There is clearly a need for a non-invasive method to deliver small andlarge therapeutic and diagnostic molecules in pharmacologicallyefficacious doses into the brain without disrupting the physiology andhomeostasis of the BBB.

SUMMARY OF THE INVENTION

The present invention relates to Insulin-Like Growth Factor 1 Receptor(IGF1R)-specific antibodies and uses thereof. More specifically, thepresent invention relates to Insulin-Like Growth Factor 1Receptor-specific antibodies and fragments thereof that transmigrate theblood-brain barrier, and uses thereof.

The present invention provides isolated or purified antibodies orfragments thereof specifically binding to an Insulin-Like Growth Factor1 Receptor (IGF1R) epitope, wherein the antibody or fragment thereoftransmigrates the blood-brain barrier, and wherein the epitope isspecifically bound by the antibody of SEQ ID NO:5. The IGF1R epitope maybe in the extracellular domain of IGF1R.

The present invention provides isolated or purified antibodies orfragments thereof comprising

-   -   a complementarity determining region (CDR) 1 sequence of        EYPSNFYA (SEQ ID NO:1);    -   a CDR2 sequence of VSRDGLTT (SEQ ID NO:2); and    -   a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:3),        wherein the antibody or fragment thereof specifically binds to        the Insulin-Like Growth Factor 1 Receptor (IGF1R).

For example, and without wishing to be limiting in any manner, theisolated or purified antibody or fragment thereof specific for IGF1R maybe

-   -   X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSNFYAMSWX₆RQAPGKX₇X₈EX₉VX₁₀G        VSRDGLTTLYADSVKGRFTX₁₁SRDNX₁₂KNTX₁₃X₁₄LQMNSX₁₅X₁₆AEDTAVYYCAIVITG        VWNKVDVNSRSYHYWGQGTX₁₇VTVSS (SEQ ID NO:4), where X₁ is E or Q;        X₂ is K or Q; X₃ iS V or E; X₄ is A or P; X₅ iS V or A; X₆ is F        or V; X₇ is E or G; X₈ is R or L; X₉ is F or W; X₁₀ is A or S;        X₁₁ is M or I; X₁₂ is A or S; X₁₃ iS V or L; X₁₄ is D or Y; X₁₅        is V or L; X₁₆ is K or R; and X₁₇ is Q or L,        or a sequence substantially identical thereto. In more specific,        non-limiting examples, the isolated or purified antibody may        comprise a sequence selected from the group consisting of:

(SEQ ID NO: 5) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS, referred to herein as IGF1R-3;(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H1;(SEQ ID NO: 7) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H2;(SEQ ID NO: 8) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKGLEFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H3;(SEQ ID NO: 9) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H4; and(SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H5,or a sequence substantially identical thereto.

The isolated or purified antibody or fragment thereof as described abovemay be a single-domain antibody (sdAb); the sdAb may be of camelidorigin.

The isolated or purified antibody or fragment thereof of the presentinvention may be presented in a multivalent display format. In amultivalent display format, the antibody or fragment thereof may belinked to a Fc fragment; the Fc fragment is the mouse Fc2b or human Fc1.For example, and without wishing to be limiting in any manner, theisolated or purified antibody or fragment thereof in multivalent displaymay comprise the sequence of SEQ ID NO:11 (referred to herein as IGF1R-3consensus-Fc fusion), SEQ ID NO:41 (referred to herein as Fc-IGF1R-3consensus fusion), or 12 (referred to herein as IGF1R-3-Fc fusion).

The isolated or purified antibody or fragment thereof as describedherein may transmigrate the blood-brain barrier.

The present invention also provides a nucleic acid molecule encoding theisolated or purified antibody or fragment thereof as described herein. Avector comprising the nucleic acid molecule as just described is alsoprovided.

The isolated or purified antibody or fragment thereof as describedherein may be immobilized onto a surface.

The present invention further provides the isolated or purified antibodyor fragment thereof as described herein linked to a cargo molecule; thecargo molecule may have a molecular weight in the range of about 1 kD toabout 200 kDa. The cargo molecule linked to the antibody or fragmentthereof may be a detectable agent, a therapeutic, a drug, a peptide, agrowth factor, a cytokine, a receptor trap, a chemical compound, acarbohydrate moiety, an enzyme, an antibody or fragments thereof, aDNA-based molecule, a viral vector, or a cytotoxic agent; one or moreliposomes or nanocarriers loaded with a detectable agent, a therapeutic,a drug, a peptide, an enzyme, and antibody or fragments thereof, aDNA-based molecule, a viral vector, or a cytotoxic agent; or one or morenanoparticle, nanowire, nanotube, or quantum dots.

Additionally, the present invention provides a composition comprisingone or more than one isolated or purified antibody or fragment thereofas described herein and a pharmaceutically-acceptable carrier, diluent,or excipient.

An in vitro method of detecting IGF1R is also provided, the methodcomprising

-   -   a) contacting a tissue sample with one or more than one isolated        or purified antibody or fragment thereof of as described herein        linked to a detectable agent; and    -   b) detecting the detectable agent detecting the detectable agent        linked to the antibody or fragment thereof bound to IGF1R in the        tissue sample.

In the method described above, the sample may be a serum sample, avascular tissue sample, tumour tissue sample, or a brain tissue samplefrom a human or animal subject. In the method as described, the step ofdetecting (steb b)) may be performed using optical imaging,immunohistochemistry, molecular diagnostic imaging, ELISA, imaging massspectrometry, or other suitable method.

Further provided is an in vivo method of detecting IGF1R expression in asubject, the method comprising:

-   -   a) administering one or more than one isolated or purified        antibody or fragment thereof as described herein linked to a        detectable agent to the subject; and    -   b) detecting the detectable agent linked to the antibody or        fragment thereof bound to IGF1R.

In the method described above, the step of detecting (steb b)) may beperformed using PET (positron emission tomography), SPECT (single-photonemission computed tomography), fluorescence imaging, or any othersuitable method.

Presently provided is a method of transporting a molecule of interestacross the blood-brain barrier (BBB), the method comprising:

-   -   a) administering one or more than one isolated or purified        antibody or fragment thereof as described herein linked to the        molecule of interest to a subject, where the antibody or        fragment thereof transmigrates the blood-brain barrier,        wherein the one or more than one antibody or fragment thereof        ferries the molecule of interest across the BBB. In the method        as just described, the molecule of interest may have a molecular        weight in the range of about 1 kD to about 200 kDa; the molecule        of interest may be a detectable agent, a therapeutic, a drug, a        peptide, a growth factor, a cytokine, a receptor trap, a        chemical compound, a carbohydrate moiety, an enzyme, an antibody        or fragment thereof, a DNA-based molecule, a viral vector, or a        cytotoxic agent; one or more liposomes or nanocarriers loaded        with a detectable agent, a therapeutic, a drug, a peptide, an        enzyme, an antibody or fragment thereof, or a cytotoxic agent;        or one or more nanoparticle, nanowire, nanotube, or quantum        dots. In the method as described, the administration may be        intravenous (iv), subcutaneous (sc), or intramuscular (im).

The invention also encompasses a method of quantifying an amount of acargo molecule delivered across the BBB of a subject, wherein the cargomolecule is linked to one or more than one isolated or purified antibodyor fragment thereof as described herein, the method comprising

-   -   a) collecting cerebrospinal fluid (CSF) from the subject; and    -   b) using targeted proteomics methods to quantify the amount of        the cargo molecule linked to one or more than one isolated or        purified antibody or fragment in the CSF.

The cargo molecule may be any desired molecule, including the cargomolecules previously described; the antibody or fragment thereoftransmigrates the BBB; the molecule may be “linked” to the antibody orfragment thereof, as previously described. In the above method, the CSFis collected from a subject using any suitable method known in the art.The amount of CSF required for targeted proteomics method in step b) maybe between about 1 to 10 μl. The targeted proteomics methods used toquantify the amount of the one or more than antibody or fragment thereoflinked to the cargo molecule may be any suitable method known in theart. For example and without wishing to be limiting, the targetedproteomics method may be a mass spectrometry method, such as multiplereaction monitoring-isotype labeled internal standards (MRM-ILIS).

The poor delivery of diagnostics or drugs across the tight and highlyselective BBB compromises the development of treatments for braindiseases, such as, but not limiting to, brain tumors andneurodegenerative diseases. The lack of carriers to transport moleculesacross the BBB delays the development of new therapeutics anddiagnostics for such diseases. As described herein, an IGF1R-bindingV_(H)H has been produced that provides an effective transport platformfor delivery of drugs conjugated to the antibody across the BBB to theirtargets in the brain. The presently described antibody exploits thenatural RMT pathway of the IGF1R from the luminal to abluminal side ofthe BBB-forming brain endothelial cells. Following binding of theantibody to IGF1R, RMT is initiated and the antibody, together with aconjugated molecule (cargo), is transcytosed through the cell to theabluminal side where they are both released into the brainmicroenvironment. The IGF1R V_(H)H was confirmed to bind to IGF1R (FIG.3C), internalize into BBB cells (FIG. 4), and cross to abluminal side ofthe in vitro BBB model (FIG. 6B). Drug-to-brain delivery studies in vivoalso showed that the IGF1R V_(H)H ‘carried’ a conjugated peptide(Galanin; about 3 kDa) as well as a large protein fusion (about 80 kDa)across the BBB (FIG. 9A and B; FIG. 6D).

The results also show that the anti-IGF1R VHH can be expressed in fusionwith Fc (fragment crystallisable) fragment to prolong circulationhalf-life by about 75 fold (about 25 h compared to about 20 min forV_(H)H alone). This high molecular weight fusion construct (about 80kDa) is also efficiently transported across the BBB. The long plasmahalf-life increases CSF exposure of the IGF1R VHH-mFc (mFc=mouse Fc)conjugate significantly compared to the V_(H)H alone and is useful as aBBB delivery carrier for the treatment of chronic diseases with targetsin the CNS. The conjugate is readily detected in brain parenchyma usingimmunofluorescence detection. The results demonstrate that the IGF1RV_(H)H carrier can “ferry” large molecules (similar in size to:antibodies, enzymes, growth factors, peptides, cytokines, receptortraps) across the BBB.

Thus, the antibody-delivery may not only be useful for short termtreatment (e.g. epileptic seizure), but may also be useful formedium-term (e.g. cancer) and long-term (e.g. Alzheimer's or Parkinson'sdisease) treatments.

Additional aspects and advantages of the present invention will beapparent in view of the following description. The detailed descriptionsand examples, while indicating preferred embodiments of the invention,are given by way of illustration only, as various changes andmodifications within the scope of the invention will become apparent tothose skilled in the art in light of the teachings of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will now be described by wayof example, with reference to the appended drawings, wherein:

FIG. 1 is a schematic diagram of the insulin-like growth factor 1receptor (IGF1R). The IGF1R is found on the cell surface, and comprisestwo subunits: the alpha subunit and the beta subunit. The alpha subunit(comprising an extracellular part with the insulin-like growth factor 1binding site) is connected by a disulphide bond to the beta subunit(comprising a small extracellular domain, a transmembrane region, and anintracellular portion). The IGF1 receptor can form a dimer. A 933 aminoacid long fragment comprising the alpha subunit and the extracellularportion of the beta subunit, as indicated within the grey box, (M1-F933,SwissProt Accession No. P08069; see FIG. 2) was recombinantly producedand used for immunization of a llama.

FIG. 2 shows the sequence of IGF1R (SwissProt Accession No. P08069 The933 amino acid long protein fragment used for immunization and panningis shown in bold; the full ectodomain is 2 amino acids longer. The furincleavage site, separating alpha and beta subunits, is shown initalicized lowercase letters. The signal peptide is shown in bolditalics.

FIG. 3A shows a size exclusion chromatogram of the IGF1R-binding V_(H)HIGF1R-3 and its humanized variants (H1, H2, H4, H5) run through aSuperdex 75 column. The profile suggests that these V_(H)H are monomericand non-aggregating. FIG. 3B shows the melting temperature (T_(m)) asdetermined by circular dichroism (CD) for IGF1R-3 V_(H)H and itshumanized variants (H1, H2, H3, H4, H5). The proteins were heated toabove 90° C. and measurements were taken in the CD to determine themelting curve and the T_(m). Subsequently, the IGF1R-3 V_(H)H was cooledto room temperature, heated once more and analysed by CD (lower curve).This allowed the determination of the fraction of refolded protein. Thiswas not carried out for the humanized versions. FIG. 3C shows a surfaceplasmon resonance (SPR) sensogram overlay for binding of 0.1-10 nMIGF1R-3 V_(H)H and its humanized variants (H1, H2, H4, H5) to therecombinant extracellular portion of the human IGF1R fragment. The datafit well to a 1:1 model. FIG. 3D shows SPR sensograms of IGF1R-3 V_(H)Hbinding to the recombinant extracellular portion of the human IGF1Rfragment in presence of IGF1. IGF1 in 100-fold excess did not affectIGF1R-3 V_(H)H binding, showing that both bind to different epitopes onthe receptor. The experiment was repeated twice. FIG. 3E shows thatIGF1R-4 V_(H)H did not bind to the recombinant extracellular portion ofthe human Insulin Receptor (IR) immobilized on SPR surface, while it didbind to the control surface of human IGF1R (IGF1R surface indicated by*asterisk). As controls, IGF1 and insulin, the natural ligands of thetwo receptors were flowed over the surfaces and binding was detected asexpected: IGF-1 bound to both receptors, while insulin binding couldonly be observed for the Insulin Receptor.

FIG. 4 shows imaging results of cell uptake of Cy-5.5-labeled IGF1R-3V_(H)H and control V_(H)H. Cy5.5-labeled IGF1R-3 V_(H)H (or FC5 V_(H)Has a positive control; Muruganandam et al., 2002; Haqqani et al., 2012)were incubated with SV40 immortalized rat brain endothelial cells(svARBEC) at 4° C. (top panels) or at 37° C. (bottom panels) to assesswhether IGF1R-3 is internalized passively (4° C.) or through activemechanisms (37° C.) such as receptor mediated endocytosis. Co-stainingwith wheat germ agglutinin and DAPI was carried out to visualize thecell surface and the nucleus, respectively. Top panel: When incubated at4° C., IGF1R-3 and FC5 V_(H)H were found outside the cells (arrowheads),bound to cell membrane (co-localized with the wheat germ agglutinin).Bottom panel: At 37° C. both FC5 and IGF1R-3 V_(H)H accumulated insidethe cells in vesicle-like structures (arrowheads), likely endosomes,suggesting internalization through an active transport mechanism.

FIG. 5A shows the sequence for the C-terminal fusion of IGF1R-3 V_(H)Hwith the murine Fc fragment. A schematic representation of the assembledfusion protein is shown in FIG. 5B with the IGF1R-3 V_(H)H shown inblack and the murine Fc (CH2 and CH3) shown in grey.

FIG. 6A is a flowchart summarizing the use of the in vitro BBB model toassess the ability of various V_(H)Hs to cross the BBB. Equimolaramounts (5.6 μM) of positive (FC5) and negative control (A20.1, aClostridium difficile toxin A-binding V_(H)H; and EG2, an EGFR bindingV_(H)H) V_(H)Hs and IGF1R-3 were tested simultaneously for their abilityto cross a rat in vitro BBB model. SV40-immortalized brain endothelialcells from adult rat (svARBECs) are grown in a monolayer on the membraneof an insert in the presence of rat astrocyte-conditioned medium in thebottom chamber and standard medium in the top chamber. Followingco-addition of equimolar amounts of the various V_(H)H to the luminalside of the BBB model, samples were taken from the bottom chamber after15, 30, and 60 min. The concentrations of each V_(H)H were thenquantified by mass spectrometry (multiple reaction monitoring-isotypelabeled internal standards; MRM-ILIS) in these samples. The P_(app)value [Qr/dt=cumulative amount in the receiver compartment versus time;A=area of the cell monolayer; C0=initial concentration of the dosingsolution] is commonly used to determine the ability of a molecule tocross the BBB. FIG. 6B shows the P_(app) values of the fourco-administered V_(H)H. IGF1R-3 has a significantly higher P_(app) valuethan FC5, while the negative controls have both very low P_(app) values,indicating facilitated transport of FC5 and IGF1R-3 V_(H)Hs compared tovery low non-specific transport or paracellular transport of controlV_(H)Hs. The results are average P_(app) values obtained in 5-6independent experiments. FIG. 6C shows the P_(app) values of thehumanized IGF1R-3 single-domain antibodies (H1, H2, H3, H4, H5) (blackbars) compared to A20.1 V_(H)H (grey bars), which was tested as acontrol in the same well (the average A20.1 value is indicated by a greydotted line). FIG. 6D shows the P_(app) values of IGF1R-3 V_(H)H andIGF1R-3-mFc (black bars), along with FC5 V_(H)H and A20.1 V_(H)H whichwere tested as controls in the same wells.

FIG. 7 shows plasma and CSF pharmacokinetics of IGF1R-3-mFc aftersystemic (tail vein) administration of 5 mg/kg. The cisterna magna wascannulated for serial CSF collections (1-48 h). Plasma and CSF levels ofIGF1R-3mFc were determined using the MRM-ILIS method that ‘tracks’ andquantifies specific protein peptide signatures. Albumin levels in theCSF were concurrently determined by MRM. All CSF samples having aplasma/CSF ratio lower than 1500 were excluded as potentiallyblood-contaminated. The plasma/CSF ratio was 0.5% for IGF1R-3-mFc 24 hafter systemic injection of the fusion protein. CSF/plasma ratio ofA20.1mFc (0.04% at 24 h) administered at the same dose (5 mg/kg)measured in prior experiments was indicated in light gray forcomparison.

FIG. 8A shows the scheme for chemical synthesis of IGF1R-3V_(H)H—Galanin conjugate. IGF1R-3 was first conjugated to the NHS groupof a Sulfo-SMCC cross-linker (1); then maleimide-activatedIGF1R-3-sulfo-SMCC was conjugated to the reduced cysteine of Galanin(2). FIG. 8B shows a SDS-PAGE gel of IGF1R-3 (lane 2), IGF1R3-SMCC (lane3), and IGF1R-3-galanin conjugate (lane 4). The ‘banding’ patternindicates the attachment of 1-2 Galanin molecules per IGF1R-3.

FIG. 9 shows IGF1R-3-mediated brain delivery of the chemicallyconjugated peptide Galanin. FIG. 9A is a graph showing the ability ofIGF1R-3 to deliver pharmacologically efficacious doses of the analgesicpeptide Galanin (3.2 kD) into the brain using Hargraeves pain model. Inthis model, localised chronic pain is induced in male Wistar rats (4-6weeks age), by injecting 100 μl of complete Freund's adjuvant (CFA) intothe left plantar surface, causing a local inflammation within a fewhours. Following tail vein injection of the BBB carrier V_(H)H-drugconjugate or Galanin alone, the rats are placed into Plexiglasenclosures set on a glass surface. A thermal stimulus is focused on theinflamed or contralateral paw via an angled mirror. The latency betweenstimulus application and paw withdrawal (lick or flick of paw) isinterpreted as a measure of the analgesic effect (inhibition of thermalhyperalgesia). The peptide Galanin alone cannot penetrate the BBB, asdemonstrated by the lack of analgesic effect after systemic (tail-vein)injection of 1 mg/kg Galanin (solid triangles). Systemic injection ofIGF1R-3-Galanin conjugate (5.85 mg/kg) induced a dose-dependentanalgesic effect over a 3 hour period, more pronounced than thatobserved with a 6mg/kg dose of FC5-Galanin conjugate. FIG. 9B showsthese results as area under the curve (AUC) of pharmacodynamics responsecompared to the maximal possible effect (MPE; control paw). 5.85 mg/kgIGF1R-3-Galanin induced 65% of MPE over a 3 hour period, demonstrating asignificant brain penetration of the conjugate compared to the Galaninalone after systemic injection (<5% of MPE). FIG. 9C shows a transientanalgesic effect induced after systemic injection of 3 mg/kg ofIGF1R-3-Galanin conjugate that disappears by 3 h after injection. Asecond injection of the same dose 1 h later produced a similar, and onlyslightly attenuated, analgesic response.

FIG. 10 shows immuno-detection of IGF1R-3mFc in brain sections 24 hafter tail-vein administration of a 6 mg/kg dose. Sacrifice perfusionwith PBS was carried out on the rats and brain sections (12 μm) wereobtained using the vibratome. IGF1R-3mFc was immuno-detected using ananti-mouse Fc antibody (red; red channel only shown in inserts). Bloodvessels in the brain section (caudate putamen, FIG. 10A; parietalcortex, FIG. 10B) were detected using lectin RCA1 (green). IGF1R-3mFccould be detected in both vessels and outside the vessels (i.e. in thebrain parenchyma, transmigrated across the BBB) as indicated byarrowheads.

FIG. 11 shows that IGF1R-3 does not interfere with insulin or IGF-1signaling through either the insulin receptor or IGF1R. FIG. 11A is arepresentative Western blot showing that neither IGF1R-3, nor any of theother anti-IGF1R VHH tested (IGF1R-1, -4, -5 or -6) induces downstreamAkt phosphorylation alone at a concentration of 100 nM. Neither does thepresence of 100 nM of IGF1R-3, or any of the other anti-IGF1R VHHsinhibit Akt phosphorylation as induced by 10 μg/ml of insulin. Thequantitation of Western blot band densities from 3 independentexperiments is shown in the bar graph (average+/−SD) below the gelimage. FIG. 11B is a representative Western blot showing that neitherIGF1R-3, nor any of the other anti-IGF1R VHH tested (IGF1R -4, -5 or -6)at 100 nM induce phosphorylation of Akt on their own and neither inhibitIGF-1 induced Akt phosphorylation (i.e. signaling) induced uponstimulation with 200 ng/ml of IGF-1. The quantitation of Western blotband densities from 3 independent experiments is shown in the bar graph(average+/−SD) below the gel image. FIG. 110 shows Western blots probedfor phosphorylated IGF1R. Cells were incubated with either 100 nM or 500nM IGF1R-3, or any of the other anti-IGF1R VHHs (IGF1R-1, -4 or -5)fused at their C-terminus to a murine Fc (e.g. IGF1R-3-mFc) alone orstimulated with 200 ng/ml IGF-1 in presence of the respectiveIGF1R-VHH-mFc fusion proteins. The Western blots indicate that none ofthe fusion constructs inhibited IGF-1 induced phosphorylation of IGF1Rand neither induced receptor phosphorylation on their own.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Insulin-Like Growth Factor 1Receptor-specific antibodies, fragments thereof, and uses thereof. Morespecifically, the present invention relates to Insulin-Like GrowthFactor 1 Receptor-specific antibodies or fragments thereof thattransmigrate the blood-brain barrier, and uses thereof.

The present invention provides isolated or purified antibodies orfragments thereof specifically binding to an Insulin-Like Growth Factor1 Receptor (IGF1R) epitope, wherein the antibody or fragment thereoftransmigrates the blood-brain barrier, and wherein the epitope isspecifically bound by the antibody of SEQ ID NO:5. The IGF1R epitope maybe in the extracellular domain of IGF1R.

The present invention provides an isolated or purified antibody orfragment thereof, comprising

-   -   a complementarity determining region (CDR) 1 sequence of        EYPSNFYA (SEQ ID NO:1);    -   a CDR2 sequence of VSRDGLTT (SEQ ID NO:2); and    -   a CDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:3),        wherein the antibody or fragment thereof specifically binds to        the Insulin-Like Growth Factor 1 Receptor (IGF1R).

The term “antibody”, also referred to in the art as “immunoglobulin”(Ig), as used herein refers to a protein constructed from paired heavyand light polypeptide chains; various Ig isotypes exist, including IgA,IgD, IgE, IgG, and IgM. When an antibody is correctly folded, each chainfolds into a number of distinct globular domains joined by more linearpolypeptide sequences. For example, the immunoglobulin light chain foldsinto a variable (V_(L)) and a constant (C_(L)) domain, while the heavychain folds into a variable (V_(H)) and three constant (C_(H), C_(H2),C_(H3)) domains. Interaction of the heavy and light chain variabledomains (V_(H) and V_(L)) results in the formation of an antigen bindingregion (Fv). Each domain has a well-established structure familiar tothose of skill in the art.

The light and heavy chain variable regions are responsible for bindingthe target antigen and can therefore show significant sequence diversitybetween antibodies. The constant regions show less sequence diversity,and are responsible for binding a number of natural proteins to elicitimportant biochemical events. The variable region of an antibodycontains the antigen-binding determinants of the molecule, and thusdetermines the specificity of an antibody for its target antigen. Themajority of sequence variability occurs in six hypervariable regions,three each per variable heavy (V_(H)) and light (V_(L)) chain; thehypervariable regions combine to form the antigen-binding site, andcontribute to binding and recognition of an antigenic determinant. Thespecificity and affinity of an antibody for its antigen is determined bythe structure of the hypervariable regions, as well as their size,shape, and chemistry of the surface they present to the antigen. Variousschemes exist for identification of the regions of hypervariability, thetwo most common being those of Kabat and of Chothia and Lesk. Kabat etat (1991) define the “complementarity-determining regions” (CDR) basedon sequence variability at the antigen-binding regions of the V_(H) andV_(L) domains. Chothia and Lesk (1987) define the “hypervariable loops”(H or L) based on the location of the structural loop regions in theV_(H) and V_(L) domains. As these individual schemes define CDR andhypervariable loop regions that are adjacent or overlapping, those ofskill in the antibody art often utilize the terms “CDR” and“hypervariable loop” interchangeably, and they may be so used herein.The CDR/loops are referred to herein according to the more recent IMGTnumbering system (Lefranc, M.-P. et al., 2003), which was developed tofacilitate comparison of variable domains. In this system, conservedamino acids (such as Cys23, Trp41, Cys104, Phe/Trp118, and a hydrophobicresidue at position 89) always have the same position. Additionally, astandardized delimitation of the framework regions (FR1: positions 1 to26; FR2: 39 to 55; FR3: 66 to 104; and FR4: 118 to 129) and of the CDR(CDR1: 27 to 38, CDR2: 56 to 65; and CDR3: 105 to 117) is provided.

An “antibody fragment” as referred to herein may include any suitableantigen-binding antibody fragment known in the art. The antibodyfragment may be a naturally-occurring antibody fragment, or may beobtained by manipulation of a naturally-occurring antibody or by usingrecombinant methods. For example, an antibody fragment may include, butis not limited to a Fv, single-chain Fv (scFv; a molecule consisting ofV_(L) and V_(H) connected with a peptide linker), Fab, F(ab′)₂, singledomain antibody (sdAb; a fragment composed of a single V_(L) or V_(H)),and multivalent presentations of any of these. Antibody fragments suchas those just described may require linker sequences, disulfide bonds,or other type of covalent bond to link different portions of thefragments; those of skill in the art will be familiar with therequirements of the different types of fragments and various approachesand various approaches for their construction.

In a non-limiting example, the antibody fragment may be an sdAb derivedfrom naturally-occurring sources. Heavy chain antibodies of camelidorigin (Hamers-Casterman et al, 1993) lack light chains and thus theirantigen binding sites consist of one domain, termed V_(H)H. sdAb havealso been observed in shark and are termed V_(NAR) (Nuttall et al,2003). Other sdAb may be engineered based on human Ig heavy and lightchain sequences (Jespers et al, 2004; To et al, 2005). As used herein,the term “sdAb” includes those sdAb directly isolated from V_(H),V_(H)H, V_(L), or V_(NAR) reservoir of any origin through phage displayor other technologies, sdAb derived from the aforementioned sdAb,recombinantly produced sdAb, as well as those sdAb generated throughfurther modification of such sdAb by humanization, affinity maturation,stabilization, solubilization, camelization, or other methods ofantibody engineering. Also encompassed by the present invention arehomologues, derivatives, or fragments that retain the antigen-bindingfunction and specificity of the sdAb.

SdAb possess desirable properties for antibody molecules, such as highthermostability, high detergent resistance, relatively high resistanceto proteases (Dumoulin et al, 2002) and high production yield(Arbabi-Ghahroudi et al, 1997); they can also be engineered to have veryhigh affinity by isolation from an immune library (Li et al, 2009) or byin vitro affinity maturation (Davies & Riechmann, 1996). Furthermodifications to increase stability, such as the introduction ofnon-canonical disulfide bonds (Hussack et al, 2011; Kim et al, 2012),may also be brought to the sdAb.

A person of skill in the art would be well-acquainted with the structureof a single-domain antibody (see, for example, 3DWT, 2P42 in ProteinData Bank). An sdAb comprises a single immunoglobulin domain thatretains the immunoglobulin fold; most notably, only threeCDR/hypervariable loops form the antigen-binding site. However, and aswould be understood by those of skill in the art, not all CDR may berequired for binding the antigen. For example, and without wishing to belimiting, one, two, or three of the CDR may contribute to binding andrecognition of the antigen by the sdAb of the present invention. The CDRof the sdAb or variable domain are referred to herein as CDR1, CDR2, andCDR3, and numbered as defined by Lefranc, M.-P. et al. (2003).

The antibody or fragment thereof of the present invention is specificfor Insulin-Like Growth Factor 1 Receptor (IGF1R), a receptor found oncell surfaces. The IGF1R comprises an alpha subunit, which comprises anextracellular part having the insulin-like growth factor 1 binding site,connected by a disulphide bond to the beta subunit, which comprises asmall extracellular domain, a transmembrane region, and an intracellularportion. The IGF1 receptor assembles into a homo-dimer or may form aheterodimer with the insulin receptor. The sequence of IGF1R may be, butis not limited to that shown in FIG. 2 (SwissProt Accession No. P08069;SEQ ID NO:13), or a sequence substantially identical thereto.

The antibody or fragment thereof as described herein should notinterfere with signaling through the Insulin Receptor (IR) or IGF1R.Specifically, the antibodies or fragments thereof as described hereinshould not inhibit AKT phosphorylation induced by insulin, nor shouldthey induce phosphorylation of the IR on their own or inhibitinsulin-induced signaling; additionally, the antibodies or fragmentsthereof described herein should not inhibit IGF-1-inducedphosphorylation of IGF1R. Moreover, they should not bind to the InsulinReceptor.

As previously stated, the antibody or fragment thereof may be an sdAb.The sdAb may be of camelid origin or derived from a camelid V_(H)H, andthus may be based on camelid framework regions; alternatively, the CDRdescribed above may be grafted onto V_(NAR), V_(H)H, V_(H) or V_(L)framework regions. In yet another alternative, the hypervariable loopsdescribed above may be grafted onto the framework regions of other typesof antibody fragments (Fv, scFv, Fab) of any source (for example, mouse)or proteins of similar size and nature onto which CDR can be grafted(for example, see Nicaise et al, 2004).

The present invention further encompasses an antibody or fragment thatis “humanized” using any suitable method known in the art, for example,but not limited to CDR grafting and veneering. Humanization of anantibody or antibody fragment comprises replacing an amino acid in thesequence with its human counterpart, as found in the human consensussequence, without loss of antigen-binding ability or specificity; thisapproach reduces immunogenicity of the antibody or fragment thereof whenintroduced into human subjects. In the process of CDR grafting, one ormore than one of the CDR defined herein may be fused or grafted to ahuman variable region (V_(H), or V_(L)), to other human antibody (IgA,IgD, IgE, IgG, and IgM), to other human antibody fragment frameworkregions (Fv, scFv, Fab) or to other proteins of similar size and natureonto which CDR can be grafted (Nicaise et al, 2004). In such a case, theconformation of said one or more than one hypervariable loop is likelypreserved, and the affinity and specificity of the sdAb for its target(i.e., IGF1R) is likely minimally affected. CDR grafting is known in theart and is described in at least the following: U.S. Pat. No. 6,180,370,U.S. Pat. No. 5,693,761, U.S. Pat. No. 6,054,297, U.S. Pat. No.5,859,205, and European Patent No. 626390. Veneering, also referred toin the art as “variable region resurfacing”, involves humanizingsolvent-exposed positions of the antibody or fragment; thus, buriednon-humanized residues, which may be important for CDR conformation, arepreserved while the potential for immunological reaction againstsolvent-exposed regions is minimized. Veneering is known in the art andis described in at least the following: U.S. Pat. No. 5,869,619, U.S.Pat. No. 5,766,886, U.S. Pat. No. 5,821,123, and European Patent No.519596. Persons of skill in the art would also be amply familiar withmethods of preparing such humanized antibody fragments and humanizingamino acid positions.

For example, and without wishing to be limiting in any manner, theisolated or purified antibody or fragment thereof specific for IGF1R maybe

-   -   X₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSNFYAMSWX₆RQAPGKX₇X₈EX₉VX₁₀G        VSRDGLTTLYADSVKGRFTX₁₁SRDNX_(i2)KNTX₁₃X₁₄LQMNSX₁₅X₁₆AEDTAVYYCAIVITG        VWNKVDVNSRSYHYWGQGTX₁₇VTVSS (SEQ ID NO:4), where X₁ is E or Q;        X₂ is K or Q; X₃ iS V or E; X₄ is A or P; X₅ iS V or A; X₆ is F        or V; X₇ is E or G; X₈ is R or L; X₉ is F or W; X₁₀ is A or S;        X₁₁ is M or I; X₁₂ is A or S; X₁₃ is V or L; X₁₄ is D or Y; X₁₅        is V or L; X₁₆ is K or R; and X₁₇ is Q or L,        or a sequence substantially identical thereto. Alternatively,        the isolated or purified antibody may comprise a sequence        selected from the group consisting of:

(SEQ ID NO: 5) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS, referred to herein as IGF1R-3;(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H1;(SEQ ID NO: 7) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H2;(SEQ ID NO: 8) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKGLEFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H3;(SEQ ID NO: 9) QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H4; and(SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS, referred to herein as IGF1R-3_H5,or a sequence substantially identical thereto.

A substantially identical sequence may comprise one or more conservativeamino acid mutations. It is known in the art that one or moreconservative amino acid mutations to a reference sequence may yield amutant peptide with no substantial change in physiological, chemical,physico-chemical or functional properties compared to the referencesequence; in such a case, the reference and mutant sequences would beconsidered “substantially identical” polypeptides. A conservative aminoacid substitution is defined herein as the substitution of an amino acidresidue for another amino acid residue with similar chemical properties(e.g. size, charge, or polarity). These conservative amino acidmutations may be made to the framework regions of the sdAb whilemaintaining the CDR sequences listed above and the overall structure ofthe CDR of the antibody or fragment; thus the specificity and binding ofthe antibody are maintained.

In a non-limiting example, a conservative mutation may be an amino acidsubstitution. Such a conservative amino acid substitution may substitutea basic, neutral, hydrophobic, or acidic amino acid for another of thesame group. By the term “basic amino acid” it is meant hydrophilic aminoacids having a side chain pK value of greater than 7, which aretypically positively charged at physiological pH. Basic amino acidsinclude histidine (His or H), arginine (Arg or R), and lysine (Lys orK). By the term “neutral amino acid” (also “polar amino acid”), it ismeant hydrophilic amino acids having a side chain that is uncharged atphysiological pH, but which has at least one bond in which the pair ofelectrons shared in common by two atoms is held more closely by one ofthe atoms. Polar amino acids include serine (Ser or S), threonine (Thror T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N),and glutamine (Gln or Q). The term “hydrophobic amino acid” (also“non-polar amino acid”) is meant to include amino acids exhibiting ahydrophobicity of greater than zero according to the normalizedconsensus hydrophobicity scale of Eisenberg (1984). Hydrophobic aminoacids include proline (Pro or P), isoleucine (Ile or I), phenylalanine(Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp orW), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G).“Acidic amino acid” refers to hydrophilic amino acids having a sidechain pK value of less than 7, which are typically negatively charged atphysiological pH. Acidic amino acids include glutamate (Glu or E), andaspartate (Asp or D).

Sequence identity is used to evaluate the similarity of two sequences;it is determined by calculating the percent of residues that are thesame when the two sequences are aligned for maximum correspondencebetween residue positions. Any known method may be used to calculatesequence identity; for example, computer software is available tocalculate sequence identity. Without wishing to be limiting, sequenceidentity can be calculated by software such as NCBI BLAST2 servicemaintained by the Swiss Institute of Bioinformatics (and as found atca.expasy.org/tools/blast/), BLAST-P, Blast-N, or FASTA-N, or any otherappropriate software that is known in the art.

The substantially identical sequences of the present invention may be atleast 90% identical; in another example, the substantially identicalsequences may be at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% identical, or any percentage therebetween, at the amino acid levelto sequences described herein. Importantly, the substantially identicalsequences retain the activity and specificity of the reference sequence.In a non-limiting embodiment, the difference in sequence identity may bedue to conservative amino acid mutation(s). In a non-limiting example,the present invention may be directed to an antibody or fragment thereofcomprising a sequence at least 95%, 98%, or 99% identical to that of theantibodies described herein.

The antibody or fragment thereof of the present invention may alsocomprise additional sequences to aid in expression, detection orpurification of a recombinant antibody or fragment thereof. Any suchsequences or tags known to those of skill in the art may be used. Forexample, and without wishing to be limiting, the antibody or fragmentthereof may comprise a targeting or signal sequence (for example, butnot limited to ompA), a detection/purification tag (for example, but notlimited to c-Myc, His₅, or His₆), or a combination thereof. In anotherexample, the additional sequence may be a biotin recognition site suchas that described by Cronan et al in WO 95/04069 or Voges et al inWO/2004/076670. As is also known to those of skill in the art, linkersequences may be used in conjunction with the additional sequences ortags, or may serve as a detection/purification tag.

The antibody or fragment thereof of the present invention may also be ina multivalent display format, also referred to herein as multivalentpresentation. Multimerization may be achieved by any suitable method ofknown in the art. For example, and without wishing to be limiting in anymanner, multimerization may be achieved using self-assembly moleculessuch as those described in Zhang et al (2004a; 2004b) and WO2003/046560,where pentabodies are produced by expressing a fusion protein comprisingthe antibody or fragment thereof of the present invention and thepentamerization domain of the B-subunit of an AB₅ toxin family (Merritt& Hol, 1995). A multimer may also be formed using the multimerizationdomains described by Zhu et al. (2010); this form, referred to herein asa “combody” form, is a fusion of the antibody or fragment of the presentinvention with a coiled-coil peptide resulting in a multimeric molecule(Zhu et al., 2010). Other forms of multivalent display are alsoencompassed by the present invention. For example, and without wishingto be limiting, the antibody or fragment thereof may be presented as adimer, a trimer, or any other suitable oligomer. This may be achieved bymethods known in the art, for example direct linking connection (Nielsonet al, 2000), c-jun/Fos interaction (de Kruif & Logtenberg, 1996), “Knobinto holes” interaction (Ridgway et al, 1996).

Another method known in the art for multimerization is to dimerize theantibody or fragment thereof using an Fc domain, for example, but notlimited to human Fc domains. The Fc domains may be selected from variousclasses including, but not limited to, IgG, IgM, or various subclassesincluding, but not limited to IgG1, IgG2, etc. In this approach, the Fcgene in inserted into a vector along with the sdAb gene to generate asdAb-Fc fusion protein (Bell et al, 2010; lqbal et al, 2010); the fusionprotein is recombinantly expressed then purified. For example, andwithout wishing to be limiting in any manner, multivalent displayformats may encompass chimeric or humanized formats of anti-IGF1R-3V_(H)H linked to an Fc domain, or bi- or tri-specific antibody fusionswith two or three anti-IGF1R-3 V_(H)H recognizing unique epitopes. Suchantibodies are easy to engineer and to produce, can greatly extend theserum half-life of sdAb, and may be excellent tumor imaging reagents(Bell et al., 2010).

The Fc domain in the multimeric complex as just described may be anysuitable Fc fragment known in the art. The Fc fragment may be from anysuitable source; for example, the Fc may be of mouse or human origin. Ina specific, non-limiting example, the Fc may be the mouse Fc2b fragmentor human Fc1 fragment (Bell et al, 2010; lqbal et al, 2010). The Fcfragment may be fused to the N-terminal or C-terminal end of theanti-IGF1R-3 V_(H)H or humanized versions of the present invention. In aspecific, non-limiting example, the multimerized isolated or purifiedantibody or fragment as just described may comprise the sequence of SEQID NO:11, 41, or 12.

Each subunit of the multimers described above may comprise the same ordifferent antibodies or fragments thereof of the present invention,which may have the same or different specificity. Additionally, themultimerization domains may be linked to the antibody or antibodyfragment using a linker, as required; such a linker should be ofsufficient length and appropriate composition to provide flexibleattachment of the two molecules, but should not hamper theantigen-binding properties of the antibody.

The antibody or fragment thereof as described herein may transmigrateacross the blood brain barrier. The brain is separated from the rest ofthe body by a specialized endothelial tissue known as the blood-brainbarrier (BBB). The endothelial cells of the BBB are connected by tightjunctions and efficiently prevent many therapeutic compounds fromentering the brain. In addition to low rates of vesicular transport, onespecific feature of the BBB is the existence of enzymatic barrier(s) andhigh level(s) of expression of ATP-dependent transporters on theabluminal (brain) side of the BBB, including P-glycoprotein (Gottesmanet al., 1993; Watanabe, 1995), which actively transport variousmolecules from the brain into the blood stream (Samuels, 1993). Onlysmall (<500 Daltons) and hydrophobic (Pardridge, 1995) molecules canmore readily cross the BBB. Thus, the ability of the antibody orfragment thereof as described above to specifically bind the surfacereceptor, internalize into brain endothelial cells, and undergotranscytosis across the BBB by evading lysosomal degradation is usefulin the neurological field.

The present invention also encompasses nucleic acid sequences encodingthe molecules as described herein. Given the degeneracy of the geneticcode, a number of nucleotide sequences would have the effect of encodingthe polypeptide, as would be readily understood by a skilled artisan.The nucleic acid sequence may be codon-optimized for expression invarious micro-organisms. The present invention also encompasses vectorscomprising the nucleic acids as just described. Furthermore, theinvention encompasses cells comprising the nucleic acid and/or vector asdescribed.

The present invention further encompasses the isolated or purifiedantibody or fragments thereof immobilized onto a surface using variousmethodologies; for example, and without wishing to be limiting, theantibody or fragment may be linked or coupled to the surface via His-tagcoupling, biotin binding, covalent binding, adsorption, and the like.Immobilization of the antibody or fragment thereof of the presentinvention may be useful in various applications for capturing, purifyingor isolating proteins. The solid surface may be any suitable surface,for example, but not limited to the well surface of a microtiter plate,channels of surface plasmon resonance (SPR) sensorchips, membranes,beads (such as magnetic-based or sepharose-based beads or otherchromatography resin), glass, plastic, stainless steel, a film, or anyother useful surface such as nanoparticles, nanowires and cantileversurfaces.

The invention also encompasses the antibody or fragment thereof asdescribed above linked to a cargo molecule. The cargo molecule may beany suitable molecule, which is delivered across the BBB by the antibodyor fragment thereof. The cargo molecule may have a molecular weight inthe range of about 1 kD to about 200 kDa; for example, the cargomolecule may have a molecular weight of about 1, 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115,120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185,190, 195, or 200 kDa, or any weight therebetween, or any range ofweights defined by any two aforementioned weights. In specific,non-limiting examples, the cargo molecule may have a molecular weight of1 kDa (for example, but not limited to a small molecule such as Cy5.5),1-10 kDa (for example, but not limited to a peptide such as galanin, 3kDa), about 80 kDa (for example, but not limited to a Fc fragment,enzyme, protein, antibody etc), or about 200 kDa (for example, but notlimited to a monoclonal antibody).

For example, and without wishing to be limiting in any manner, the cargomolecule may be a detectable agent, a therapeutic agent, a drug, apeptide, an enzyme, a growth factor, a cytokine, a receptor trap, anantibody or fragment thereof (e.g., IgG, scFv, Fab, V_(H)H, V_(H),V_(L), etc) a chemical compound, a carbohydrate moiety, DNA-basedmolecules (anti-sense oligonucleotide, microRNA, siRNA, plasmid), acytotoxic agent, viral vector (adeno-, lenti-, retro), one or moreliposomes or nanocarriers loaded with any of the previously recitedtypes of cargo molecules, or one or more nanoparticle, nanowire,nanotube, or quantum dots. The cargo molecule as described above may bea detectable agent. For example, the IGF1R-specific antibody or fragmentthereof may be linked to a radioisotope, a paramagnetic label, afluorophore, a fluorescent agent, Near Infra-Red (NIR; for exampleCy5.5) fluorochrome or dye, an echogenic microbubble, an affinity label,a detectable protein-based molecule, nucleotide, quantum dot,nanoparticle, nanowire, or nanotube or any other suitable agent that maybe detected by imaging methods. The antibody or fragment thereof may belinked to the cargo molecule using any method known in the art(recombinant technology, chemical conjugation, etc.).

The cargo molecule as described herein may be linked, also referred toherein as “conjugated”, to the antibody or fragment thereof by anysuitable method known in the art. For example, and without wishing to belimiting, the cargo molecule may be linked to the peptide by a covalentbond or ionic interaction. The linkage may be achieved through achemical cross-linking reaction, or through fusion using recombinant DNAmethodology combined with any peptide expression system, such asbacteria, yeast or mammalian cell-based systems. When conjugating thecargo molecule to the antibody or fragment thereof, a suitable linkermay be used. Methods for linking an antibody or fragment thereof to acargo molecule such as a therapeutic or detectable agent would bewell-known to a person of skill in the art.

In one non-limiting example, the cargo molecule may be a detectablelabel, a radioisotope, a paramagnetic label such as gadolinium or ironoxide, a fluorophore, Near Infra-Red (NIR) fluorochrome or dye, anechogenic microbubble, an affinity label (for example biotin, avidin,etc), enzymes, or any other suitable agent that may be detected bydiagnostic imaging methods. In a specific, non-limiting example, theanti-IGF1R-3 or fragment thereof may be linked to a near infraredfluorescence (NIRF) imaging dye, for example and not wishing to belimiting Cy5.5, Alexa680, Dylight680, or Dylight800.

Thus, the present invention further provides an in vitro method ofdetecting IGF1R, comprising contacting a tissue sample with one or morethan one isolated or purified antibody or fragment thereof of thepresent invention linked to a detectable agent. The IGF1R-antibodycomplex can then be detected using detection and/or imaging technologiesknown in the art. The tissue sample in the method as just described maybe any suitable tissue sample, for example but not limited to a serumsample, a vascular tissue sample, a tumour tissue sample, or a braintissue sample; the tissue sample may be from a human or animal subject.The step of contacting is done under suitable conditions, known to thoseskilled in the art, for formation of a complex between the antibody orfragment thereof and IGF1R. The step of detecting may be accomplished byany suitable method known in the art, for example, but not limited tooptical imaging, immunohistochemistry, molecular diagnostic imaging,ELISA, imaging mass spectrometry, or other suitable method. For example,and without wishing to be limiting in any manner, the isolated orpurified antibody or fragment thereof linked to a detectable agent maybe used in immunoassays (IA) including, but not limited to enzyme IA(EIA), ELISA, “rapid antigen capture”, “rapid chromatographic IA”, and“rapid EIA”. (For example, see Planche et al, 2008; Sloan et al, 2008;Rüssmann et al, 2007; Musher et al, 2007; Turgeon et al, 2003; Fenner etal, 2008).

The present invention also provides an in vivo method of detecting IGF1Rexpression in a subject. The method comprises administering one or morethan one isolated or purified antibody or fragment thereof as describedherein linked to a detectable agent to the subject, then detecting thelabelled antibody or fragment thereof bound to IGF1R. The step ofdetecting may comprise any suitable method known in the art, forexample, but not limited to PET, SPECT, or fluorescence imaging, or anyother suitable method. The method as just described may be useful indetecting the expression of IGF1R in blood vessels or tissues, forexample but not limited to tumor tissues.

The in vivo detection step in the methods described above may be wholebody imaging for diagnostic purposes or local imaging at specific sites,such as but not limited to brain vessels or brain tumor vessels, in aquantitative manner to assess the progression of disease or hostresponse to a treatment regimen. The detection step in the methods asdescribed above may be immunohistochemistry, or a non-invasive(molecular) diagnostic imaging technology including, but not limited to:

-   -   Optical imaging;    -   Positron emission tomography (PET), wherein the detectable agent        is an isotopes such as ¹¹C, ¹³N, ¹⁵O, ¹⁸F, ⁶⁴Cu, ⁶²Cu, 1²⁴I,        ⁷⁶Br, ⁸²Rb and ⁶⁸Ga, with ¹⁸F being the most clinically        utilized;    -   Single photon emission computed tomography (SPECT), wherein the        detectable agent is a radiotracer such as ^(99m)Tc, ¹¹¹In, ¹²³I,        ²⁰¹Tl, ¹³³Xe, depending on the specific application;    -   Magnetic resonance imaging (MRI), wherein the detectable agent        may be, for example and not limited to gadolinium, iron oxide        nanoparticles and carbon-coated iron-cobalt nanoparticles        thereby increasing the sensitivity of MRI for the detection of        plaques.    -   Contrast-Enhanced Ultrasonography (CEUS) or ultrasound, wherein        the detectable agent is at least one acoustically active and        gas-filled microbubble. Ultrasound is a widespread technology        for the screening and early detection of human diseases. It is        less expensive than MRI or scintigraphy and safer than molecular        imaging modalities such as radionuclide imaging because it does        not involve radiation.

The present invention further provides a method of transporting amolecule of interest across the blood-brain barrier. The methodcomprises administering the molecule linked to an antibody or fragmentthereof as described herein to a subject; the antibody or fragmentthereof transmigrates the blood-brain barrier. The molecule may be anydesired molecule, including the cargo molecules as previously described;the molecule may be “linked” to the antibody or fragment thereof usingany suitable method, including, but not limited to conjugation orexpression in a fusion protein. The administration may be by anysuitable method, for example parenteral administration, including butnot limited to intravenous (iv), subcutaneous (sc), and intramuscular(im) administration. In this method, the antibody or fragment thereof ofthe present invention ‘ferries’ the molecule of interest across the BBBto its brain target.

The present invention also encompasses a composition comprising one ormore than one isolated or purified antibody or fragment thereof asdescribed herein. The composition may comprise a single antibody orfragment as described above, or may be a mixture of antibodies orfragments. Furthermore, in a composition comprising a mixture ofantibodies or fragments of the present invention, the antibodies mayhave the same specificity, or may differ in their specificities; forexample, and without wishing to be limiting in any manner, thecomposition may comprise antibodies or fragments thereof specific toIGF1R (same or different epitope).

The composition may also comprise a pharmaceutically acceptable diluent,excipient, or carrier. The diluent, excipient, or carrier may be anysuitable diluent, excipient, or carrier known in the art, and must becompatible with other ingredients in the composition, with the method ofdelivery of the composition, and is not deleterious to the recipient ofthe composition. The composition may be in any suitable form; forexample, the composition may be provided in suspension form, powder form(for example, but limited to lyophilised or encapsulated), capsule ortablet form. For example, and without wishing to be limiting, when thecomposition is provided in suspension form, the carrier may comprisewater, saline, a suitable buffer, or additives to improve solubilityand/or stability; reconstitution to produce the suspension is effectedin a buffer at a suitable pH to ensure the viability of the antibody orfragment thereof. Dry powders may also include additives to improvestability and/or carriers to increase bulk/volume; for example, andwithout wishing to be limiting, the dry powder composition may comprisesucrose or trehalose. In a specific, non-limiting example, thecomposition may be so formulated as to deliver the antibody or fragmentthereof to the gastrointestinal tract of the subject. Thus, thecomposition may comprise encapsulation, time-release, or other suitabletechnologies for delivery of the antibody or fragment thereof. It wouldbe within the competency of a person of skill in the art to preparesuitable compositions comprising the present compounds.

The invention also encompasses a method of quantifying an amount of acargo molecule delivered across the BBB of a subject, wherein the cargomolecule is linked to one or more than one isolated or purified antibodyor fragment thereof as described herein, the method comprising

-   -   c) collecting cerebrospinal fluid (CSF) from the subject; and    -   d) using targeted proteomics methods to quantify the amount of        the cargo molecule linked to one or more than one antibody or        fragment thereof in the CSF.

The cargo molecule may be any desired molecule, including the cargomolecules, as previously described; the isolated or purified antibody orfragment thereof transmigrates the blood-brain barrier; and the moleculemay be “linked” to the antibody or fragment thereof using any suitablemethod, including, but not limited to conjugation or expression in afusion protein, as previously described. In the above method, the CSF iscollected from a subject using any suitable method known in the art. Theamount of CSF required for targeted proteomics method in step b) may bebetween about 1 to 10 μl; for example, the amount of CSF required may beabout 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,7.5, 8.0, 8.5, 9.0, 9.5, or 10 μl, or any amount therebetween, or anyrange defined by the amount just described. The antibody or fragmentlinked to the cargo molecule may have been administered to the subjectprior to collection of the CSF. A suitable delay between administrationand delivery of the antibody or fragment linked to the cargo moleculeacross the BBB may be required. The delay may be at least 30 minutesfollowing administration of the antibody or fragment linked to the cargomolecule; for example and without wishing to be limiting in any manner,the delay may be at least 30 minutes, 1 hour, 1.5 hour, 2 hours, 2.5hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours. The targetedproteomics methods used to quantify the amount of the one or more thanantibody or fragment thereof linked to the cargo molecule may be anysuitable method known in the art. For example and without wishing to belimiting, the targeted proteomics method may be a mass spectrometrymethod, such as but not limited to multiple reaction monitoring using anisotopically labeled internal standard (MRM-ILIS; see for exampleHaqqani et al., 2013). MRM is advantageous in that it allows for rapid,sensitive, and specific quantification of unlabelled targeted analytes(for example, an antibody or fragment thereof as described herein) in abiological sample. The multiplexing capability of the assay may allowfor quantification of both the antibody or fragment thereof and thecargo molecule.

The present invention will be further illustrated in the followingexamples. However, it is to be understood that these examples are forillustrative purposes only and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLE 1 Purification of IGF1R Recombinant Fragment

A 933 amino acid long recombinant fragment of the extracellular domainof IGF1R (shown by the grey box in FIG. 1; see also amino acids 1-933 ofSEQ ID NO:13) was prepared. The fragment comprised an N-terminal 30amino acid signal peptide, the full alpha subunit, a furin cleavage site(RKRR, SEQ ID NO:14; separating alpha and beta subunits), as well as themajority of the extracellular portion of the beta subunit (FIGS. 1 and2).

Cloning. The sequence of the IGF1R ectodomain of interest was amplifiedby PCR using the following primers:

(forward; SEQ ID NO: 15) 5′-CGGGATCCGCCACCATGAAGTCTGGCTCCGGAG-3′(reverse; SEQ ID NO: 16) 5′-GCTCTAGATCAGAAGTTTTCATATCCTGTTTTGG-3′and subcloned into the SmaI site of Puc19. The IGF1R⁹³³ sequence wasthen sub-cloned into pCDN4/myc-His (Invitrogen) to generatepIGF1R⁹³³-His, which allows for expression of a His-tagged ectodomain asdescribed previously (Samani et al. 2004).

Transient transfection. Lentivirus particles expressing IGF1R⁹³³-Hiswere generated in the packaging cell line 293SF-PacLV, as detailedpreviously (Broussau et al., 2008). Briefly, the cells were transfectedwith the vector using polyethylenimine. Fresh medium (LC-SFM),containing 1 μg/ml doxycycline and 10 μg/ml cumate, was added to thecells 5 hours post-transfection and the supernatants containing LVparticles were collected after 48-72 hours, concentrated byultracentrifugation at 100,000×g for 2 h at 4° C. on a 20% sucrosecushion (Gaillet B et al. 2007), re-suspended in LC-SFM mediumsupplemented with 1% FBS and stored at −70° C. until used.

Stable expression. A stable cell line, 293SF-cum2-CR5-IGF1R-His, wasgenerated by transduction of 293SF-Cum2 cell lines with the respectivelentivirus particles using a protocol described previously (Gaillet B.et al. 2010). Briefly, 0.5-1.0×10⁵ 293SF-Cum2 cells were seeded in 24wells plates into 200 μl of LC-SFM medium without dextran sulfate. TheLV suspension was prepared by mixing 200-500 μL of LV with 8 μg/mL ofpolybrene and incubating it for 30 min at 37° C. The freshly made LVsuspension was added to the cells 4 hours after seeding. After 24 h, 500μL medium supplemented with dextran sulfate was added to the cells. Toincrease the level of expression the cells were re-transduced up to 6times using the same protocol after 3-4 days of cell recovery. Finallythe cells were expanded in 6-well plates and shaker flasks.

Large scale protein production and purification. The clone identified asthe highest producer was expanded in shaker or spinner flasks. Proteinproduction was initiated by the addition of 1 μg/ml cumate in freshmedium, followed by a 24 h incubation at 37° C. and a 4-8 day incubationat 30° C. Cells were removed by centrifugation and the supernatantsfiltered and concentrated (10×) using the Tangential Flow FiltrationSystems (Pellicon ultrafiltration cassettes, EMD Millipore).

The IGF1R⁹³³-His was purified using a HisPrep column (GE Healthcare)according to the manufacturer's instructions. Briefly, the concentratedsample was applied to a His-prep FF (16/10) column (GE Healtcare),equilibrated and washed with 50 mM sodium phosphate, 300 mM NaCl, 5 mMimidazole pH 7.5 and eluted with the 50 mM sodium phosphate, 300 mMNaCl, 500 mM imidazole pH 7.5. A step elution with 0.1 M sodium citratepH 4.5 to pH 2.5 was used to elute the protein and peak fractions werepooled. Buffer exchange was performed by ultrafiltration using a 50 kDacut-off membrane or a desalting column with a buffer containing 50 mMsodium phosphate, 150 mM NaCl and 0.01% Tween-80, pH 7.2. Purity of bothproteins was verified by SDS-PAGE and they were stored at −80° C. untilused (see subsequent examples).

EXAMPLE 2 Llama Immunization and Serum Response

To isolate V_(H)Hs that targets the extracellular domain of IGF1R, allama was immunized with the recombinant IGF1R⁹³³-His fragment obtainedin Example 1.

One male llama (Lama glama) was immunized by sub-cutaneous, lower-backinjection of IGF1R⁹³³-His recombinant antigen (Example 1). On day 1, 200μg of antigen diluted in PBS to 1 ml was injected together with 1 ml ofFreund's Complete Adjuvant (Sigma, St. Louis, Mo.). Three furtherinjections of 100 μg of IGF1R⁹³³-His antigen plus Freund's IncompleteAdjuvant (Sigma) were performed on days 22, 36, and 50. A finalinjection of 100 μg of antigen with no adjuvant was performed on day 77.Pre-immune blood was drawn before the first injection on day 1 andserved as a negative control. Blood (10-15 ml) was collected on days 29,43, 57 and 84. The blood from day 84 was processed immediately toisolate peripheral blood mononuclear cells (PBMC). The blood was diluted1:1 with phosphate buffered saline (PBS) and PBMCs were purified fromthe blood using the Lymphoprep Tube (Axis Shield). The cells werecounted and stored as aliquots of about 1×10⁷ cells at −80° C. forfuture use.

Pre-immune and post-immune total serum was analyzed for a specificresponse to IGF1R⁹³³-His antigen by ELISA on day 57. Llama sera from day84 were fractionated as previously described (Doyle et al, 2008). Theresulting fractions, A1 (HCAb), A2 (HCAb), G1 (HCAb) and G2 (clgG) wereanalyzed for specific binding to the IGF1R⁹³³-His antigen by ELISA.Briefly, 5 μg of IGF1R⁹³³-His recombinant antigen diluted in PBS wasincubated overnight (100 μl/well, 18 h, 4° C.) in 96 well Maxisorpplates (Nalgene, Nunc) to coat the plates. Plates were blocked withbovine serum albumin (BSA), washed with PBS-T (PBS+0.05% (v/v)Tween-20), and serial dilutions of pre-immune total serum, post-immunetotal serum (day 57), and fractionated serum (day 84) were applied.After incubation at room temperature for 1.5 h the plates were washedwith PBS-T, before goat anti-llama IgG (1:1,000 in PBS) was added andplates were incubated for 1 h at 37° C. After washing with PBS-T, piganti-goat IgG-HRP conjugate (1:3,000 in PBS) was added and plates wereincubated for 1 h at 37° C. A final PBS-T wash was carried out prior tothe addition of 100 μl/well TMB substrate (KPL, Gaithersburg, Md.); thesubstrate was incubated for 10 min. The reaction was stopped with 100dal/well 1 M H₃PO₄. Absorbance was read at 450 nm.

EXAMPLE 3 Library Construction and Selection of IGF1R-Binding V_(H)Hs

A hyperimmunized llama V_(H)H library was constructed based on RNAisolated from the PBMCs in Example 2.

Library construction and panning was performed essentially as previouslydescribed (Arbabi-Ghahroudi et al, 2009a, 2009b; Tanha et al, 2003).Total RNA was isolated from approximately 10⁷ PBMCs collected on day 84post-immunization (Example 2) using the QlAamp RNA Blood Mini Kit(Qiagen). About 5 μg of total RNA was used as template for first strandcDNA synthesis with oligo dT primers using the First-Strand cDNASynthesis Kit (GE Healthcare). The cDNA was amplified by an equimolarmix of three variable region-specific sense primers:

MJ1: (SEQ ID NO: 17) 5′-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3′ MJ2: (SEQ ID NO: 18)5′-GCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA- 3′ MJ3:(SEQ ID NO: 19) 5′-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGTGGAGTCT- 3′,and two antisense CH₂-specific primers:

(SEQ ID NO: 20) CH₂: 5′-CGCCATCAAGGTACCAGTTGA-3′ (SEQ ID NO: 21) CH₂b₃:5′-GGGGTACCTGTCATCCACGGACCAGCTGA-3′.

Briefly, the PCR reaction mixture was set up in a total volume of 50 μlwith the following components: 1-3 μl cDNA, 5 pmol of MJ1-3 primermixture, 5 pmol of CH₂ or CH₂b₃ primers, 5 μl of 10× reaction buffer, 1μl of 10 mM dNTP, 2.5 unit of Taq DNA polymerase (Hoffmann-La Roche).The PCR protocol consisted of an (i) initial step at 94° C. for 3 min,(ii) followed by 30 cycles of 94° C. for 1 min, 55° C. for 30 s, 72° C.for 30 s and (iii) a final extension step at 72° C. for 7 min. Theamplified PCR products were run on a 2% agarose gel and two major bandswere observed: a band of about 850 bp, corresponding to conventionalIgG, and a second band of around 600 bp, corresponding to the V_(H)H—CH2region of camelid heavy chain antibodies. The smaller bands were cut andpurified using the QIAquick Gel Extraction Kit (Qiagen) and re-amplifiedin a second PCR in a total volume of 50 μl using 1 μl (30 ng) of DNAtemplate, 5 pmol of each of MJ7 primer (5′-CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC-3′ SEQ ID NO:22) and MJ8 primer(5′-CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG-3′ SEQ ID NO:23), 5μl of 10× reaction buffer, 1 μl of 10 mM dNTP, 2.5 unit of Taq DNApolymerase. The PCR protocol consisted of (i) an initial step at 94° C.for 3 min, (ii) followed by 30 cycles of 94° C. for 30 s, 57° C. for 30s and 72° C. for 1 min and (iii) a final extension step at 72° C. for 7min. The amplified PCR products, ranging between 340 bp and 420 bp andcorresponding to V_(H)H fragments of heavy chain antibodies, werepurified using the QIAquick PCR Purification Kit (Qiagen), digested withSfiI restriction enzyme (New England BioLabs) and re-purified using thesame kit.

80 μg of pMED1 phagemid vector (Arbabi-Ghahroudi et al, 2009b) weredigested with SfiI overnight at 50° C. To minimize self-ligation, 20units of XhoI and PstI restriction enzymes were added to cut the excisedfragment and the digestion reaction was incubated for an additional 2 hat 37° C. 60 μg of digested phagemid DNA was ligated with 6 μg ofdigested (SfiI for 5 h at 50° C.) V_(H)H fragments (molar ratio 1:1) for3 h at room temperature using LigaFast Rapid DNA Ligation System(Promega) according to the manufacturer's instructions. The ligatedplasmids were purified using the QIAquick PCR Purification Kit (Qiagen),eluted in a final volume of 100 μl, and transformed intoelectrocompetent TG1 E. coli (Stratagene) using 5 μl of ligated DNAaliquot per transformation reaction, as described (Arbabi-Ghahroudi etal, 2009b). The size of the library was determined to be 5×10⁷ asdescribed in (Arbabi-Ghahroudi et al, 2009b). 20 clones were sequencedand contained all unique V_(H)H sequences. The E. coli, containing thelibrary was grown for 2-3 h at 37° C., 250 rpm in the presence of 2%(w/v) glucose. The bacteria were then pelleted, resuspended in 2×YT/Amp/Glu (2× YT medium with 100 μg/ml ampicillin and 2% (w/v) glucose)with 35% (v/v) glycerol and stored at −80° C. in small aliquots.

Panning experiments were essentially performed as described in (Arbabiet al, 1997). Two milliliters of the library (2.0×10¹⁰ bacteria) werethawed on ice and grown in 2× YT/Amp/Glu for about 2 h at 37° C.(A₆₀₀=0.4-0.5). The E. coli were subsequently infected with 20× excessM13KO7 helper phage (New England Biolabs) for 1 h at 37° C. The culturewas then centrifuged at 4° C. and infected bacterial pellets werere-suspended in 200 ml of 2× YT/Amp with 50 μg/ml kanamycin andincubated at 37° C. and 250 rpm. The phage particles in the culturesupernatant were incubated with ⅕ volume of 20% PEG 8000/2.5M NaCl onice for 1 h and centrifuged at 10,000 rpm for 15 min. The phage pelletswere re-suspended in 1.5 ml of sterile PBS, titrated and used as inputphage for panning. For panning round 1, 96-well Maxisorp™ plates werecoated with 10 μg of recombinant IGF1R⁹³³-His per well in 100 μl PBSovernight at 4° C. The wells were rinsed with PBS and blocked with PBSplus 1% (w/v) casein for 2 h at 37° C. Approximately 10¹² phages wereadded to the blocked wells and incubated for 2 h at 37° C. After 10×washing with PBS/0.1% (v/v) Tween 20, the bound phages were eluted with0.1 M triethylamine, neutralized (50 μl of 1M Tris-HCl, pH 7.4) andmixed with exponentially growing TG1 E. coli. Titration of eluted phagewas performed and infected bacteria were superinfected with M13K07 andgrown overnight at 37° C. The purified phage from the overnight culturewas used as the input for the next round of panning. The panning wascontinued for three further rounds. The same protocol as described abovewas used, except that the amount of recombinant antigen used to coat theplates was reduced to 7 μg, 5 μg and 5 μg for the second, third andfourth rounds of panning, respectively.

Individual TG1 colonies obtained after round four of panning weresubjected to phage ELISA screening, essentially as described elsewhere(Doyle et al, 2008), with the exception that 5 μg/ml of IGF1R⁹³³-Hisrecombinant antigen were used to coat the microtiter plates. Allpositive clones were sent for DNA sequencing. Unique clones that gavehigh phage ELISA signals were selected for large-scale expression andpurification using known methods (see Example 4). A clone dubbed IGF1R-3was identified for further study; its sequence is shown below.

(SEQ ID NO: 3) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS

EXAMPLE 4 Humanization of IGF1R-3

To avoid potential immunogenicity of llama-derived IGF1R-3 when appliedas BBB carrier for therapeutics, to the camelid-derived sdAb was“humanized” by mutation of “camelid” residues in the V_(H)H. It shouldbe noted that, for the purpose of humanization, Kabat numbering (Kabatet al, 1991) was used for identification of CDR residues.

3D-structure modeling of camelid V_(H)Hs. Template structures similar toIGF1R-3 V_(H)H were identified using BLAST searches against the ProteinData Bank (PDB). The 3D structure of the IGF1R-3 was approximated usinghomology modeling based on 4KRP|B (PDB code|Chain ID) as the maintemplate, with additional information from 4FHB|D. The IGF1R-3 V_(H)Hstructure was then built by mutating the main template structure to theIGF1R-3 sequence; this included 35 mutations at various positions. TheIGF1R-3 V_(H)H model was then refined by energy minimization with theAMBER force-field and a stepwise release of constraints, ranging fromthe CDR loops, which were relaxed first, to the backbone heavy atoms ofthe framework region, which were fully relaxed only in the last stage.The CDR-H3 loop of the V_(H)H model was then refined byMonte-Carlo-minimization (MCM) conformational sampling, in whichdihedral angles in the CDR-H3 region were sampled followed by energyminimization.

Selection of the human heavy-chain framework for the camelid CDR. Humanheavy-chain framework was selected by standard sequence homologycomparison against the human germline databases (VBASE), against othersequence databases (Genbank and SwissProt), and against the humanframework consensus sequences. BLAST searches were conducted to retrievesequence matches with highest homology in the framework region only(i.e., excluding CDR) while matching the length of the CDR. The closesthuman frameworks identified for IGF1R-3 V_(H)H corresponded to the humanVH-3 subgroup. Several human germline VH-3 framework sequences that weremost similar to IGF1R-3 V_(H)H were also retained in addition to thehuman VH-3 consensus sequence. The IGF1R-3 V_(H)H framework sequencesrequired 18 mutations in order to arrive at the consensus human VH-3sequence for 100% framework humanization.

Identification of framework residues for back-mutations. The IGF1R-3V_(H)H model and its fully-humanized counterpart were characterized toestimate the humanness index, antigen contact propensity index, todelineate the CDR, canonical residues, unusual framework residues,potential glycosylation sites, buried residues, Vernier zone residues,and proximity to CDR. The analysis of these data suggested the design ofseveral humanized variants for the anti-IGF1R V_(H)H, each varianthaving varying numbers of back-mutations to the parent camelid residuesat various positions. 5 humanized variants were designed for IGF1R-3V_(H)H (IGF1R-3_H1 to IGF1R-3_H5), where variants contained up to 10back-mutations. Some of these camelid back-mutations residues wereburied inside the V_(H)H domain core and hence are not expected toinduce an immune response.

EXAMPLE 5 Expression and Purification of selected V_(H)H Constructs

IGF1R-3 identified in Example 3 and the humanized versions constructedin Example 4 (collectively referred to herein as “V_(H)H constructs”)were sub-cloned into expression plasmids for protein expression andpurification.

A pPhagemid vector containing the DNA of the IGF1R-3 V_(H)H constructwas purified using the MiniPrep Kit (Qiagen). The IGF1R-binding V_(H)HIGF1R-3 was PCR amplified from the pMED1 phagemid vector, adding anN-terminal Bbsl cleavage site and a BamHl cleavage site at theC-terminus, using primers:

(forward; SEQ ID NO: 24) 5′-TATGAAGACACCAGGCCCAGGTAAAGCTGGAGGAGTCT-3′(reverse; SEQ ID NO: 25) 5′-TTGTTCGGATCCTGAGGAGACGGTGACCTG-3′

The PCR fragment and the pSJF2H expression vector was digested with Bbsland BamHl restriction enzymes (NEB) according to the manufacturer'sinstructions. Following digestion, each digested IGF1R-3 V_(H)H fragmentwas ligated into the digested pSJF2H expression vector, using methodssimilar to those described in Arbabi-Ghahroudi et al. (2009b); theligation products were then transformed into electro-competent TG1 E.coli. Clones were selected on LB agar plates+100 μg/ml ampicillin andverified by DNA sequencing.

The humanized clones were synthesized and directly cloned into pSJF2H,similarly as described above and subsequently transformed into E. coliTG1 and selected as described above.

Protein Expression. All IGF1R-3 V_(H)H was expressed in TG1 E. coli. Anovernight culture in LB/amp/glu medium (LB medium supplemented with 100μg/ml ampicillin and 1% glucose) was subcultured at 1:100 dilution in 1L LB/amp/glu. Protein expression was induced at an OD₆₀₀ of 0.8-0.9 bythe addition of IPTG to a final concentration of 0.2 mM. The culture wasgrown at 220 rpm overnight at 37° C. The bacteria were pelleted bycentrifuging at 6000 rpm for 12 min; the pellet was re-suspended in 35ml of cold TES buffer (0.2M Tris-Cl pH 8.0, 20% sucrose, 0.5 mM EDTA).The suspension was incubated on ice and vortexed every 10 min for 1hour. Then 45 ml of cold TES (⅛ volume of total volume) was added andimmediately vortexed for 1 minute and for 15 seconds every 10 minthereafter for 1 hour to extract the protein from the periplasm. Theresulting supernatant containing V_(H)H was filtered through a 0.22 μmmembrane and dialysed overnight into immobilized metal-affinitychromatography (IMAC) buffer A (10 mM HEPES pH 7.0, 500 mM NaCl). Theprotein was purified using HiTrap Chelating HP columns (GE Healthcare)as described previously (Arbabi-Ghahroudi 2009b). Eluted proteinfractions were analyzed by SDS-PAGE and Western blotting before beingdialysed against PBS as described previously (Arbabi-Ghahroudi 2009b).The purified protein fractions were pooled and dialyzed against PBS+3 mMEDTA, and the protein concentration was determined.

EXAMPLE 6 Biophysical Characterization of Anti-IGF1R V_(H)H IGF1R-3

The anti-IGF1R V_(H)H IGF1R-3 constructs expressed and purified inExample 4 were characterized using size exclusion chromatography,melting temperature analysis, and surface plasmon resonance analysis

Size exclusion chromatography: Size exclusion chromatography employingSuperdex™ 75 (GE Healthcare) was used to eliminate any possibleaggregates prior to Surface Plasmon Resonance (SPR) analysis. Therunning buffer used was 10 mM HEPES, pH7.4 containing 150 mM NaCl, 3 mMEDTA and 0.005% P20. Concentrations of fractions used for SPR analysiswere determined by measuring absorbance at 280 nm wavelength. The SECanalysis suggested that IGF1R3 V_(H)H and its humanized variants H1, H2,H4 and H5 were monomeric, based on the elution volume compared tostandards (FIG. 3A).

Melting temperature: The thermal stability of the IGF1R-3 V_(H)H andhumanized constructs was evaluated using melting temperature (T_(m))measurement by CD spectroscopy. A Jasco J-815 spectropolarimeterequipped with a Peltier thermoelectric type temperature control system(Jasco, Easton, Md., USA) was used to carry out experiments. A CDcuvette with a path length of 1 mm was used. The spectra were recordedover a wavelength range of 180-260 nm with scanning speed of 50 nm/min,digital integration time (DIT) of 4 s, a band width of 1 nm, data pitchof 1 nm, and an integration time of 1 s. To measure melting temperatureor T_(m) (Greenfield, 2006a; 2006b), CD spectra were recorded over atemperature range of 30° C. to 96° C. All CD spectra were subtractedfrom the blank corresponding to buffer spectra. Measurements wereperformed with concentrations of 50 μg/mL V_(H)H in 100 mM sodiumphosphate buffer, pH 7.4. Heat-induced protein denaturation wasmonitored at 210 nm for all variants. The fraction folded (ff) wasobtained by a formula as described (Greenfield, 2006a; 2006b):

ff=([θ]_(T)−[θ]_(U))/([θ]_(F)−[θ]_(U))   formula I

where [θ]_(T) is the molar ellipticity at any temperature, [θ]_(F), isthe molar ellipticity of the fully folded protein at 30° C. and [θ]_(U)is the molar ellipticity of the unfolded protein at 90° C. Meltingtemperature (T_(m)) was obtained as a midpoint of the unfolding curve(fraction folded (ff) versus temperature) by a nonlinear regressioncurve fit (Boltzman sigmoidal equation) using the graphing softwareGraphPad Prism (version 4.02 for Windows). The melting temperatures(T_(m)) of V_(H)H were determined based on ellipticity data assuming atwo-state system, which is in agreement with the observed denaturationcurves corresponding to a sharp transition into denaturation. The T_(m)values were taken at midpoint of the sigmoidal denaturation curves offraction folded (ff) versus temperature. Results are shown in FIG. 3B.The melting temperatures of most humanized V_(H)H were improved (higher)in comparison to the IGF1R-3 V_(H)H, suggesting improved biophysicalproperties.

Surface Plasmon Resonance (SPR): The binding of monomeric IGF1R-3 V_(H)Hconstructs to immobilized recombinant human IGF1R (Example 1) wasdetermined by SPR using BIACORE 3000 (GE Healthcare). Approximately 3000Resonance Units (RU) of recombinant human IGF1R were immobilized on aSensor chip CM5. Immobilization was carried out at a concentration of 10μg/ml in 10 mM acetate at pH4.0 using the amine coupling kit supplied bythe manufacturer. The remaining binding sites were blocked with 1 Methanolamine pH 8.5. An ethanolamine blocked surface was used as areference surface. For the binding studies, analyses were carried out at25° C. in 10 mM HEPES, pH7.4 containing 150 mM NaCl, 3 mM EDTA and0.005% surfactant P20 (Polyoxyethylenesorbitan; GE Healthcare). Variousconcentrations of the IGF1R-4 V_(H)H were injected over the immobilizedhuman IGF1R or Insulin receptor (IR) and reference surfaces at a flowrate of 20 μl/min. Surfaces were regenerated with 10 mM glycine pH 2.0with a contact time of 24 seconds. Data were analyzed with BIAevaluation4.1 software (GE Healthcare). The sensograms in FIG. 3C show that thedata fit well to a 1:1 model, giving K_(D) and ‘off-rates’ shown inTable 1. This indicates that IGF1R-3 V_(H)H and the humanized variantsare high-affinity single-domain antibodies binding to extracellulardomain of human and rat IGF1R.

TABLE 1 Affinity of IGF1R-3 constructs for human IGF1R as determined bysurface plasmon resonance. K_(D) (nM) k_(d) (s⁻¹) IGF1R-3 1.3 1.3 × 10⁻⁴IGF1R-3 H1 47 2.3 × 10⁻³ IGF1R-3 H2 6.6 7.2 × 10⁻⁴ IGF1R-3 H4 1.4 5.5 ×10⁻⁴ IGF1R-3 H5 7.5 2.5 × 10⁻³

The SPR analyses were further used to demonstrate that IGF1R-3 V_(H)Hdoes not bind to the same epitope on the receptor as the natural ligandIGF-1 (FIG. 3D). The experiment was set up, carried out and analysed asdescribed above. Binding to freshly immobilized human IGF1R surface wasstudied by injection of human IGF1 at a concentration of 25×K_(D)followed by co-injection of IGF1R-3 both at concentrations 25×K_(D),with flow rates of 20 μl/min and injection times of 5 minutes. Surfaceswere regenerated by washing with running buffer. Data was analyzed asdescribed above. The natural ligand IGF-1 bound the receptor withsaturation reached at 70RU; the IGF1R-3 V_(H)H bound to the IGF1R-IGF-1complex with the expected ˜265RU (relative units; binding saturation).The simultaneous binding of both IGF1R-3 V_(H)H and IGF-1 to thereceptor demonstrates that both bind to different epitopes.

The cross-reactivity of IGF1R-3 V_(H)H binding to human Insulin Receptorwas also evaluated using SPR. The experiment was set up, carried out andanalysed as described above. Briefly, besides human IGF1R, approximately4000 Resonance Units (RU) of recombinant human Insulin Receptor (R&Dsystems) was immobilized on a separate cell on a Sensor chip CM5.Binding to freshly immobilized human Insulin Receptor and IGF1R wasanalysed by injecting IGF1R-3 VHH (1 nM), Insulin (100 nM) and humanIGF1 (100 nM) at flow rates of 20 μl/min and injection times of 1minute. Surfaces were regenerated by washing with running buffer. Whilebinding to IGF1R could be observed (FIG. 3E marked by asterisk *), nobinding to the Insulin Receptor surface was observed, suggesting thatIGF1R-3 cannot bind the Insulin receptor. As controls, IGF1 and insulin,the natural ligands of the two receptors were flowed over the surfacesand binding was detected as expected: IGF-1 bound to both receptors,while insulin binding could only be observed for the Insulin Receptor.

EXAMPLE 7 Internalization of the IGF1R-3 by Brain Endothelial Cells

To determine whether IGF1R-3 is internalized into cells, svARBEC cellswere incubated with Cy5-5-labelled IGF1R-3.

IGF1R-3 V_(H)H was labeled with NHS-Cy5.5. The labeling was done througha stable amine bond between primary amines (at the N-terminus and/or onlysine side chains of the protein) and the NHS ester. Typically, 10% v/vof 1M carbonate/bicarbonate buffer (759 mM bicarbonate, 258 mMcarbonate) pH 9.3 was added to 4 mg of V_(H)H prepared in PBS (1.06 mMKH₂PO₄, 154 mM NaCl, 5.6 mM Na₂HPO₄) pH7.4 and adjusted to a finalconcentration of 4 mg/mL. The NHS-Cy5.5, dissolved in DMSO at 10 mg/mL,was added at a 2× molar ratio of dye to protein. The mixture wasincubated at room temperature for 2 h with several inversions in a 1.5mL microcentrifuge tube. Following the incubation, unbound dye andreaction bi-products were filtered using Zeba Spin Desalting Columns, 7KMWCO (Pierce) and measured using a Beckman DU530 spectrophotometer(Beckman Coulter). Cy5.5-labeled IGF1R-3 or FC5 as a positive control (1mg/ml) were incubated with SV40 immortalized rat brain endothelial cells(svARBEC) at 4° C. (FIG. 4, top panels), thus allowing only for passive,non-specific transport mechanism to occur, or at 3TC (FIG. 4, bottompanels) to allow for active transport such as receptor mediatedendocytosis to take place. Co-staining with wheat germ agglutinin andDAPI was carried out to visualize the cell surface and the nucleus,respectively. Cells were observed under fluorescent microscope andimages were captured.

If incubated at 4° C., IGF1R-3 was found outside the cells co-localizingwith the cell membrane stained with wheat germ agglutinin. In contrast,when incubated at 37° C., IGF1R-3 accumulated in vesicles inside thecells, likely endosomes, suggesting that the antibody internalized intocells through an active transport mechanism. Similar behaviour wasobserved for FC5, previously shown to enter the cells byenergy-dependent endocytosis via clathrin-coated vesicles (Abulrob etal.2005).

EXAMPLE 8 Production of a IGF1R-3-mFc Construct

A construct comprising IGF1R-3 V_(H)H fused to a murine antibodyfragment crystallisable (Fc; mFc2b) was prepared, expressed, andisolated. The sequence of the C-terminal IGF1R-3-mFc construct is shownin FIG. 5A, with a schematic of the molecule shown in FIG. 5B. Thefusion protein (˜80 kDa) also comprised a N-terminal signal peptide(MEFGLSWVFLVAILKGVQC; SEQ ID NO:40) that is not shown in the sequence ofFIG. 5A.

The IGF1R-3 cDNA was cloned into mammalian expression vector pTT5(Durocher, 2002) containing the mouse Fc2b fragment. Polyplexes of theresulting vector were pre-formed by mixing 25 ml of plasmid DNA solutioncontaining 187.5 μg pTT5-IR5mFc2b, 56.25 μg pTT-AKTdd (activated mutantof Protein Kinase B), 18.75 μg pTTo-GFP (to monitor transfectionefficiency), and 112.5 μg of salmon testis DNA (Sigma-Aldrich); and 25ml of PEI solution containing 1.125 mg of PEIpro™ (PolyPlusTransfection), both made in F17 medium. The mixture was incubated for 10minutes prior to addition to the cell culture. A 450 ml culture of CHOcells stably expressing a truncated EBNA1 protein (CHO-3E7) and grown inF17 medium (Invitrogen) was transfected with 50 ml of polyplexes. Twentyfour hours post-transfection, the culture was fed with 12.5 ml of 40%(w/v) tryptone N1 (Organotechnie) solution and 1.25 ml of 200 mMvalproic acid solution. The culture was harvested 8 dayspost-transfection and clarified by centrifugation. Clarified medium wasfiltered through a 0.22 μm membrane prior to its application on a columnpacked with 5 ml of protein-A MabSelect SuRe resin (GE Healthcare).After loading, the column was washed with 5 volumes ofphosphate-buffered saline pH 7.1 (PBS) and the antibody was eluted with100 mM sodium citrate buffer pH 3.0. Fractions containing the elutedantibody were pooled and a buffer exchange was performed by loading on adesalting Econo-Pac column (BioRad) equilibrated in PBS. Desaltedantibody was then sterile-filtered by passing through a Millex GP(Millipore) filter unit (0.22 μm) and aliquoted.

EXAMPLE 9 Transport of the IGF1R-3 and IGF1R-3mFc Across in Vitro BloodBrain Barrier Model

To evaluate whether the IGF1R-3 V_(H)H and the construct of Example 8transmigrate the blood-brain barrier, an in vitro assay was used asdescribed below. A flow chart summarizing the experiment is shown atFIG. 6A.

SV40-immortalized Adult Rat Brain Endothelial Cells (Sv-ARBEC) were usedto generate an in vitro blood-brain barrier (BBB) model as described(Garberg et al., 2005; Haqqani et al., 2012). Sv-ARBEC (80,000cells/membrane) were seeded on a 0.1 mg/mL rat tail collagen typeI-coated tissue culture inserts (pore size-1 μm; surface area 0.9 cm²,Falcon) in 1 ml of growth medium. The bottom chamber of the insertassembly contained 2 ml of growth medium supplemented with theimmortalized neonatal rat astrocytes-conditioned medium in a 1:1 (v/v)ratio. Equimolar amounts (5.6 μM) of positive (FC5) or negative controls(A20.1, a Clostridium difficile toxin A binding V_(H)H; and EG2, an EGFRbinding V_(H)H) and IGF1R-3 were tested for their ability to cross thisrat in vitro BBB model. Following exposure of equimolar amounts of thesdAbs to the luminal side of the BBB, samples were taken after 15, 30,and 60 min from the abluminal side. The sdAb content of each sample wasthen quantified by mass spectrometry (multiple reactionmonitoring-isotype labeled internal standards; MRM-ILIS) as described byHaqqani et al. (2012) (see method description below).

Determination of the apparent permeability coefficient: Quantifiedvalues can be directly plotted or the P_(app) (apparent permeabilitycoefficient) values can be determined with the given formula (FIG. 6A)and plotted. The P_(app) value is commonly used to determine the abilityof a molecule to cross the BBB. [Qr/dt=cumulative amount in the receivercompartment versus time; A=area of the cell monolayer; CO=initialconcentration of the dosing solution]. P_(app) values are a measure ofthe specific permeability of the compound across brain endothelialmonolayer.

Results are shown in FIGS. 6B-D. The results given are average P_(app)values obtained from several independent experiments. Both negativecontrols have a very low P_(app) value, indicating that non-specifictransport or paracellular transport of these V_(H)Hs across the BBBmodel is minimal. IGF1R-3 V_(H)H has a high P_(app) value, indicatinghigh rate of transport across the in vitro BBB model. The P_(app) forIGF1R-3 V_(H)H is approximately 3-fold higher than that of a positivecontrol—BBB-permeable V_(H)H FC5 (WO 02/057445). The results providestrong indication that IGF1R-3 undergoes a facilitated trans-cellulartransport across brain endothelial cells in vitro and could have similarproperties in vivo. Humanized IGF1R-3 V_(H)H variants H1, H2, H3 and H4had 20-30% reduced P_(app) values compared to IGF1R3 V_(H)H, whereasvariant H5 showed similar P_(app) values to IGF1R-3 V_(H)H (FIG. 6C).

The P_(app) value for IGF1R-3mFc (FIG. 6D) was significantly reducedcompared to IGF1R V_(H)H, however still 2.5-fold higher than P_(app)value of positive control antibody, FC5 (FIG. 6D). The data suggest thateither the linkage orientation of IGF1R-3 to Fc or bi-valent formatreduces its BBB-crossing ability compared to monomeric IGF1R-3. It isworth noting that constructs comprising IGF1R-5 or a humanized versionlinked to a cargo molecule (MW ˜110 kDa or 180 kDa) have also been shownto be ferried across the BBB (data not shown).

Absolute quantitation of V_(H)H using MRM-ILIS method. The methods areall as described in Haqqani et al. (2012). Briefly, to develop the SRM(selected reaction monitoring also known as multiple reaction monitoring(MRM) assay for V_(H)H, each V_(H)H was first analyzed by nanoLC-MS/MSusing data-dependent acquisition to identify all ionizible peptides. Foreach peptide, the 3 to 5 most intense fragment ions were chosen. Aninitial SRM assay was developed to monitor these fragments at attomoleamounts of the digest (about 100-300 amol). Fragments that showedreproducible intensity ratios at low amounts (i.e., had Pearson r2≧0.95compared to higher amounts) were considered stable and were chosen forthe final SRM assay. To further optimize the assay, elution times foreach peptide were also included, with care taken to not choose peptidesthat have close m/z (mass-to-charge ratio) and elution times.

A typical multiplexed SRM analysis of V_(H)H in cell media or bodyfluids (serum or cerebrospinal fluid (CSF)) involved spiking knownamount of ILIS (0.1-10 nM) followed by injecting 100-400 ng of CSF orcultured media proteins (0.3-1 μL) or about 50-100 ng of serum proteins(1-3 nanoliters) into the nanoLC-MS system. The precursor m/z of eachtarget peptide ion was selected in the ion trap (and the remainingunrelated ions were discarded) at the specified elution time for thetarget, followed by collision induced dissociation (CID) fragmentation,and selection of only the desired fragment ions in the ion trap formonitoring by the detector. For quantification analysis, raw filesgenerated by the LTQ (ThermoFisher) were converted to the standard massspectrometry data format mzXML and intensities were extracted using anin-house software called Q-MRM (Quantitative-MRM; see Haqqani et al.2012), which is a modified version of MatchRx software. For each V_(H)H,extracted-ion chromatograms were generated for each of its fragment ionthat consisted of combined intensities within 0.25 Da of the fragmentm/z over the entire elution time. To obtain a final intensity value foreach fragment, all intensities within 0.5 min of the expected retentiontimes were summed. A V_(H)H was defined as detectable in a sample if thefragments of at least one of its peptides showed the expected intensityratios, i.e., the final intensity values showed a strong Pearsoncorrelation r≧4.95 and p<0.05 compared with the final intensities valuesof its corresponding pure V_(H)H.

Samples containing mixtures of V_(H)H (media, serum, CSF) were reduced,alkylated and trypsin-digested as previously described (Haqqani et al.,2012; Gergov et al., 2003). The digests (tryptic peptides) wereacidified with acetic acid (5% final concentration) and analyzed on areversed-phase nanoAcquity UPLC (Waters, Milford, Mass.) coupled to LTQXL ETD or LTQ Orbitrap ETD mass spectrometer (ThermoFisher, Waltham,Mass.). The desired aliquot of the sample was injected and loaded onto a300 μm I.D.×0.5 mm 3 μm PepMaps C18 trap (ThermoFisher) then eluted ontoa 100 μm I.D.×10 cm 1.7 μm BEH130C18 nanoLC column (Waters) using agradient from 0%-20% acetonitrile (in 0.1% formic) in 1 minute, 20%-46%in 16 min, and 46%-95% in 1 min at a flow rate of 400 nL/min. The elutedpeptides were ionized into the mass spectrometer by electrosprayionization (ESI) for MS/MS and SRM analysis using CID for fragmentationof the peptide ions. The CID was performed with helium as collision gasat normalized collision energy of 35% and 30 ms of activation time. Ioninjection times into linear ion trap were adjusted by the instrumentusing an automatic gain control (AGC) target value of 6×10³ and amaximum accumulation time of 200 ms

The V_(H)H -specific peptides used for detection and quantification ofeach V_(H)H in multiplexed assay are shown in Table 2.

TABLE 2 Peptides used in nanoLC-SRM detection of FC5,FC5-ILIS, EG2, A20.1, IGF-1R-5 and albumin.(a) In various studies described, assays weremultiplexed in different combinations forsimultaneous monitoring in the same sample;(b) Heavy-labeled peptide; (c) Limits of de-tection and quantification of the SRM assayfor each peptide ranged from 1.5-2.5 ng/ml.1 ng/mL corresponds to about 60-70 pM of V_(H)H.A20-1 as described in Hussack et al, 2011b;EG2 as described in Iqbal et al, 2010. SEQ ID Protein Signatures NO:Unique IGF1R3 EFVAGVSR 26 Yes LSCVASEYPSNFYAMSWFR 27 Yes NTVDLQMNSVK 28Yes SYHYWGQGTQVTVSSGSEQK 29 Yes FC5 ITWGGDNTFYSNSVK 30 Yes FC5-ITWGGDNTFYSNSVK^((b)) 30 Yes ILIS A20.1 TTYYADSVK 31 Yes EFVAAGSSTGR 32Yes TFSMDPMAWFR 33 Yes DEYAYWGQGTQVTVSSGQAGQGSEQK 34 Yes EG2 DFSDYVMGWFR35 Yes LEESGGGLVQAGDSLR 36 Yes NMVYLQMNSLKPEDTAVYYCAVNSAGTYVSPR 37 YesAlbumin APQVSTPTLVEAAR 38 Yes

EXAMPLE 10 IGF1R-3-mFc Levels in CSF and Plasma

An in vivo assay was carried out to determine whether IGF1R-3-mFc(Example 8) is able to cross into the brain, and specifically into thecerebrospinal fluid (CSF), as well as to quantify its presence in CSFand serum.

Animals were housed singly in polypropylene cages, and were allowed freeaccess to food and water. Experiments were done in a 12 h light/darkcycle at a temperature of 24° C. and a relative humidity of 50±5%. Allanimal procedures were approved by the NRC's Animal Care Committee andwere in compliance with the Canadian Council of Animal Care guidelines.

Male Wistar rats, 8-10 weeks of age (weight range, 230-250 g) were used.To sample CSF, the fur on the neck and head region of animals was shavedand they were then placed in a Plexiglas chamber and moderatelyanesthetized with 3% isoflurane; the CSF was collected essentially asdescribed by Nirogi et al (2009). The anesthetized rat was placed in ametal frame instrument (generously provided by Dr. VinicioGranados-Soto; CINVESTAV, Mexico) and immobilised using earbars. Theposition of the animal's head was maintained downward at approximately45°. A depressible surface with the appearance of a rhomb between theoccipital protuberance and the spine of the atlas was made visible byrubbing the cotton embedded in ethanol (75%) over this surface. 27Gneedle covered with PE-10 tubing (Becton Dickinson, Mississauga, ON,Canada) 10 cm in length and connected to a 100 cc insulin syringe wasinserted horizontally and centrally into the cisterna magna for CSFcollection without making incisions. Two resistance points (clicks)along the needle path can be easily felt, due to the tearing of the skinand the ripping of atlanto-occipital membrane. When the needle passedthe second resistance point, CSF was collected (40-100 μL) through theneedle by applying a gentle suction of the insulin syringe. After theCSF sampling, corresponding blood was collected by cardiac punctureafter thoracotomy and placed in vacutainer tubes (Becton Dickinson,Mississauga, ON, Canada) with clot activator and gel, and thencentrifuged at 3,000×g for 15 min. Serum was removed using amicropipette and rapidly frozen at −80° C. until further analyses.

Serum and CSF were collected 24 h after intravenous injection of 6mg/kgof IGF1R-3mFc or A20.1mFc. Serum and CSF samples were analyzed by massspectrometry and nanoLC-SRM based quantification as described in Example7.

CSF collection is a delicate procedure during which CSF can be easilycontaminated with blood. Since the amounts of V_(H)H are expected to bemuch smaller in the CSF (<0.1%) than blood, even a slight contaminationwith blood could seriously compromise the value of an individual CSFsample. It was therefore necessary to develop stringent exclusioncriteria for blood-contaminated CSF samples. To evaluate blood-CSFalbumin ratio, a nanoLC-SRM method was developed for quantifying albuminlevels in plasma and CSF. An albumin peptide APQVSTPTLVEAAR (SEQ IDNO:38) was selected based on its unique retention time and m/z value(Mol Pharm) in order to have minimum interference with other peptidepeaks in the multiplex assay. The intensity of the peptide wasquantified in both CSF and plasma samples using SRM as described above.The albumin ratio was calculated as follows for each rat:

Albumin Ratio=Intensity per nL of plasma analyzed/Intensity per nL ofCSF analyzed

A ratio of 1500 and below was considered as blood contaminated.

Results are shown in FIG. 7. The figure shows that CSF levels ofIGF1R-3Fc are 0.5% of serum levels compared to 0.04% of control antibodyA20.1Fc. Negative control A20.1Fc (75 kD) shows serum/CSF ratio at 24 hsimilar to those described in the literature for molecules of thesimilar size. Typical serum/CSF (lumbar) ratio for albumin (60 kD) atsteady state is 0.1% whereas serum/CSF ratio for IgG is 0.07 (Shen etal., 2004; Lin, 2008). IGF1R3-Fc serum/CSF ratio at 24 h is 11-foldhigher than that for A20.1mFc.

Additional constructs of ˜110 kDa have also been shown to be ferriedacross the BBB by 1GF1R-3 or a humanized version (data not shown).

EXAMPLE 11 Conjugation of IGF1R-3 to Galanin

To determine whether IGF1R-3 V_(H)H can cross the blood-brain barrier(BBB) in vivo and ‘ferry’ across the BBB a molecule that cannot crossthe BBB on its own, the neuropeptide Galanin was chemically conjugatedto IGF1R-3 V_(H)H and administered systemically. Galanin is aneuroactive peptide that produces analgesia by binding GaIR1 and GaIR2expressed in brain tissue. When given peripherally, Galanin has noanalgesic effects because it cannot cross the BBB on its own (Robertsonet al., 2011).

The IGF1R-3 V_(H)H was conjugated to a rat Galanin (Gal) fragment withcysteamide modified C-terminus (Biomatic)(GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-cysteamide, SEQ ID NO:39). The scheme forconjugation is shown in FIG. 8A.

Briefly, 5 mg of IGF1R-3 V_(H)H (Example 4) in 0.5× PBS, 2.5 mM EDTA at[2 mg/ml] were mixed with 436.4 μl of 2.5 mg/mlsulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC) (7.5× excess molar ratio). The mixture was then flushedwith nitrogen gas and incubated for 30 minutes at room temperature (RT)to allow the NHS ester arm of the Sulfo-SMCC to react with amines on theV_(H)H. Subsequently the unreacted Sulfo-SMCC was removed from themaleimide-activated IGF1R-5 V_(H)H using a 10 ml 7K Zeba column(Pierce). Prior to sample loading, the column was washed 3 times with 5ml PBS and spun at 1000×g for 2 min. After sample loading, the columnwas topped-up with 200 μl of PBS and was spun for 2 min at 1000'g. Asfor the IGF1R-Fc constructs, 5 mg were reacted with −68 μl of Sulfo-SMCC(6.5× excess molar ratio) in the same way as described above.

Separately and concurrently, cysteamide modified C-TERM galanin(Gal-cya) was prepared by dissolving 10 mg of lyophilized powder in 10ml of endotoxin free water to make a 1 mg/ml stock (the galanin-cyapowder has a small amount of DTT to prevent disulfide bridge formationduring purification). Finally, 100 μl of 0.5M EDTA was added (5 mM finalconcentration).

The purified maleimide-activated IGF1R-3 V_(H)H (2.6 ml) was diluted to5 ml with 0.5× PBS, 2.5 mM EDTA and then 5 ml Gal-cya was added whilevortexing. The samples were flushed with nitrogen, sealed and incubatedovernight at 4° C. The next day, the unreacted Gal-cya was removed usingAmicon-15 10K and 30K column (Millipore) respectively. The sample wereadded to the column and spun at 4000×g for 7 minutes until the volumewas reduced to 5 ml. 5 ml of 0.5× PBS, 2.5 mM EDTA was added to theremaining 5 ml sample in the column's insert and was spun again untilthe sample was reduced to 4 ml. The conjugated samples were then addedto a 10 ml 7K Zeba column (Pierce), prepared as described above, andthen spun for 2 min at 1000×g.

Conjugated IGF1R-3-Gal samples were then ran on a 16% or 10% SDS-PAGEnon-reducing gel and silver stained to confirm shift in molecular weightsize after conjugation. The reaction was titrated to achieve ˜1 to 2galanin molecules per V_(H)H. Results confirm the galanin load on theIGF1R-3 V_(H)H (see FIG. 8B).

Endotoxin removal and determination of endotoxin levels: Endotoxins wereremoved using Amicon Ultra—molecular weight cut-off (MWCO) cellulosemembrane spin columns (Millipore). First, 15 ml of the V_(H)Hpreparation was passed through an Amicon-15-50K MWCO column bycentrifugation at 4000×g for 10 minutes; the elution was collect. Thiselution was then added to an Amicon-15-10K MWCO column and spun at4000×g for 7-10 minutes resulting in a reduction of the supernatantvolume from 15 ml to 7.5 ml. The supernatant volume in the column wasadjusted back to 15 ml by adding PBS. The column was spun again asdescribed above. The supernatant was collect and the endotoxin levelswere measured with EndoSafe-PTS system using cartridges with asensitivity range of 10-0.1 EU/ml (Charles River LaboratoriesInternational). 25 ul of sample was loaded in each of the 4 wells on thecartridge and diluted if necessary. Only samples with EU<1 per 1 mg wereused for animal studies.

EXAMPLE 12 Transport of the IGF1R-3-Gal Using the Hargreaves Model

To evaluate whether the IGF1R-3-Gal (Example 11) transmigrates theblood-brain barrier, an in vivo assay, was utilized, previouslydescribed in International Patent Publication No. WO 2011/127580.

A rat model of inflammatory hyperalgesia, similar to that described byHargreaves et al. (1988), was used. Animals were housed in groups ofthree (Hargreaves model) per polypropylene cage, and were allowed freeaccess to food and water. Experiments were done in a 12 h light/darkcycle at a temperature of 24° C. and a relative humidity of 50±5%. Allanimal procedures were approved by the NRC's Animal Care Committee andwere in compliance with the Canadian Council of Animal Care guidelines.

In this model, male Wistar rats, 6-8 weeks (weight range 230-250 g) oldwere injected with low volume (100 μl with a 30-gauge needle) ofcomplete Freund's adjuvant (CFA; heat-killed M. tuberculosis (Sigma)suspended in oil:saline 1:1 emulsion) into the right hind paw underbrief isoflurane anesthesia (3%). CFA induces the release ofpro-inflammatory substances that activate nociceptors and create achronic pain state and hyperalgesia (a heightened sensitivity to noxiousheat). The paw withdrawal latency was measured by the application of aradiant stimulus in the plantar surface in both hind paws (inflamed annon-inflamed control paw) using the plantar Analgesia Meter equipmentfor paw stimulation (IITC Model #336TG Life Science, Inc.). The timetaken by the animal to respond by licking or flicking its paw wasinterpreted as positive response (paw withdrawal latency). Thelight-intensity lamp was adjusted to elicit baseline paw withdrawallatencies between 17 to 20 s in both hind paws before CFAadministration. If a withdrawal response does not occur within 20 s, thelight beam was automatically turned off to avoid tissue damage and thepaw was assigned the maximum score.

Two days post-CFA injection and prior to the administration of thecompounds, the animals were manipulated and acclimatized in theanalgesia meter equipment for at least 60 min with the aim to reducestress and prevent false positive responses The baseline was measured inboth paws to verify the developed pain (thermal hyperalgesia); thenon-inflamed paw was used as a control against the injected paw. Animalswith paw withdrawal latency of more than 6 s for the “inflamed paw” andless than 17 s for the “normal paw” were excluded from the experiment.

To determine whether IGF1R-3-Gal is delivered across the blood brainbarrier and can engage target receptors (GaIR1 and 2) in brainparenchyma, the rats received one tail vein injection of IGF1R-3-Galanin(2.93 mg/kg or 5.85 mg/kg; endotoxin EU<1) or control compounds threedays post-CFA injection. The paw withdrawal latency (PWL) was tested foreach hind paw (inflamed and non-inflamed) every 15 min for 3 hours. Anincreased latency of paw withdrawal indicates successfully induction ofanalgesia, which can only be obtained through successful delivery ofGalanin into the brain parenchyma by IGF1R-3. Galanin can only induceanalgesia when present in the brain parenchyma and on its own cannotcross an intact BBB (Robertson et al., 2011).

Results were analyzed as temporal courses of Paw Withdrawal Latencies(PWL, sec) versus time (min or hrs) (FIG. 9A). FIG. 9B shows the sameresults as area under the curve (AUC) and compares it to the % ofMaximum Possible Effect (% MPE). FIG. 9C shows that repeated injectionof the same dose of IGF1R3-Galanin, 1 hour after the analgesic responsefrom the first injection has ended, produces a similar analgesicresponse as the first injection.

The results show that intravenously administered galanin does not reducepain compared to PBS. In contrast, a single injection of FC5-Gal orIGF1R3-Gal produce measurable analgesic effect, suggesting that thisV_(H)H ‘ferries’ Galanin across the BBB to produce an analgesic effectby binding to GaIR1 and/or 2 in the brain parenchyma. The IGF1R-3-Galeffect is dose-dependent and significantly more pronounced than thatinduced by FC5-Gal, suggesting that the IGF1R receptor has a higher rateof BBB transport than the putative FC5 receptor. The repeated dosingproduces similar analgesic response, suggesting fast ‘turnover’(capacity) of the carrier receptor IGF1R. The results demonstrate thatIGF1R-3 V_(H)H can ‘ferry’ molecules of at least 3000 Da across the BBBusing receptor-mediated transcytosis pathway (the combined MW ofantibody-peptide conjugate is about 18 kDa). Active, receptor mediatedtransport is required since the BBB is known to prevent the passage ofall hydrophilic molecules greater than 0.5 kDa.

EXAMPLE 13 lmmunodetection of IGF1R-3-mFc

To ascertain that high levels of IGF1R-3Fc detected in the CSF afterperipheral administration originate at least in part from parenchymalextracellular space, in other words, that the intact construct hadcrossed the BBB, immunodetection of IGF1R-3-mFc in rat brains wasperformed.

Briefly, brains of animals were harvested immediately following animalperfusion with PBS 24 h after 6 mg/kg tail-vein injection of eitherIGF1R-3-mFc or A20.1mFc. The brains were frozen and sectioned oncryotome into 12 μm sections. Sections were fixed for 10 min RT in 100%

Methanol, washed 3× in PBS and incubated for 1 h in 10% normal goatserum (NGS) containing 0.3% TritonX-100 in 1× PBS. Goat anti-m-IgGFcy-cy3 (Cat#115-165-071, Jackson Immuno Reasearch, lot#106360) 1:200 in5% NGS containing 0.3% TritonX-100 in 1× PBS was applied overnight at 4°C. Sections were washed three times in 1× PBS Vasculature-staininglectin RCAI (Cat#FL-1081, Vector) 1:500 in 1× PBS was then added for 10min. After washing three times with 1× PBS, sections were covered with acover slip in Dako fluorescent mounting medium (Cat#S3023, Dako) andspiked with 2 μg/mL Hoechst (Cat#H3570, Invitrogen) to stain nuclei.Images were captured with Olympus 1×81 Fluorescent Microscope using 10×and 60× Objectives and channels as shown in Table 3.

TABLE 3 Objectives and channels used for fluorescent microscopy.Fluorescent molecule Excitation (nm) Emmision (nm) RCAI-vessels FITC 495518 Hoechst33342-nuclei Hoechst 350 461 IGF1R-3-m-Fc Cy3 531 593

Results are shown in FIG. 10. Immunodetection of mouse Fc showed strongstaining of brain vessels throughout different brain regions, as well asstaining of perivascular brain parenchyma, indicating that the IGF1R-3Fcis accumulated in brain vessels and also transmigrated the BBB intosurrounding brain parenchyma. In contrast, no mFc-specific stainingcould be detected in A20.1mFc-injected animals. The results support theassertion that increased CSF levels of IGF1R-3Fc are indicative of theconstruct transmigration across the BBB. This is further stronglysupported by the observation that galanin linked to IGF1R-3 inducedpharmacological response (analgesia) on parenchymal GaIR1 and GaIR2receptors. Collectively, in vitro BBB transmigration results, in vivopharmacokinetic (serum/CSF levels) and pharmacodynamic (Hargreavesmodel) results demonstrate that IGF1R-3 V_(H)H transmigrates the intactBBB at significantly higher rates than other V_(H)Hs via activereceptor-mediated transcytosis triggered by its binding to IGF1Repitopes and that it can ‘ferry’ a range (1-80 kD) of otherwisenon-permeable molecules across the blood-brain barrier.

EXAMPLE 14 IGF1R-3 Effect on ‘Physiological’ Function of IGF1R

From a safety perspective, it is important to show that the antibody ofthe invention does not interfere with the physiological function of thereceptor—i.e., signaling induced by its natural ligand, IGF-1—whenengaging its receptor for drug delivery via receptor-mediatedtranscytosis. In view of this, it is important to demonstrate thatIGF1R-3 V_(H)H or IGF1R-3-mFc do not interfere with physiologicalsignaling through IGF1R or the related insulin receptor (IR) induced bytheir natural ligands.

To determine whether IGF1R-3 induces signaling through IGF1R or IRalone, or interferes with signalling as stimulated by the receptor'snatural ligands, IGF-1 or insulin, their effect on phosphorylation ofthe receptors themselves or receptor-stimulated downstream kinase, Akt,was determined in SV-ARBEC cells.

SV-ARBEC were grown to confluence in M199 base medium supplemented withpeptone, D-glucose, BME amino acids, BME vitamins,antibiotic/antimycotic solution and fetal bovine serum, according toart-known methods. The cells were switched into serum free medium 18 hprior to treatment. IGF1R-3 VHH or IGF1R-3-Fc fusion (100 nM or 500 nM)was added to the cells 1 h prior to the addition of either 200 ng/mlIGF-1, 10 μg/ml of insulin or vehicle. The cells were incubated withligands or vehicle for 20 minutes and then washed twice in Hank'sbalanced salt solution. Cells were subsequently lysed using 1× RIPAbuffer (Cell Signaling Technology) supplemented with 1% Triton-x 100 andprotease inhibitor cocktail (Sigma). The cells were given 2×20 secondbursts in a water bath sonicator and lysates were clarified bycentrifugation at 14,000rpm for 10 minutes. Protein concentration wasdetermined using DC protein assay system (BIO-RAD laboratories). Equalμg of protein samples were resolved on a 4-20% gradient SDSpolyacrylamide gel at 125V and transferred to PVDF membrane. Phospho-Akt(Ser 473) was detected by overnight incubation in 1:1000 dilution of theprimary antibody against this target (Cell Signaling Technology)followed by a 1 h incubation with goat anti-rabbit IgG-HRP secondaryantibody then reacted with ECL Plus reagent and visualized onautoradiography film. Densitometry values were determined usingUn-Scan-It software (Silk Scientific Inc.).

The results are shown in FIG. 11. Western blot analyses of Aktphosphorylation showed that IGF1R-3 did not inhibit Akt phosphorylationinduced by 10 μg/ml of insulin or by 200 ng/ml of IGF-1 when co-appliedwith at 100 nM of IGF1R-3 or IGF1R-3-mFc or 500 nM IGF1R-3-mFc. Neitherdid either of the V_(H)H or Fc fusions induce Akt signalling on its own(FIGS. 11A, 11B and 11C, labelled “−5”). The results demonstrate thateven in bivalent display in the Fc fusion format IGF1R-3 does nottrigger receptor dimerization and down-stream signaling, and thereforedoes not interfere with the receptor function in the presence of thenatural ligand. This feature of IGF1R-3 (‘silent binder’) is importantfor its application as a BBB carrier for therapeutics, since it confersa favourable safety profile.

The embodiments and examples described herein are illustrative and arenot meant to limit the scope of the invention as claimed. Variations ofthe foregoing embodiments, including alternatives, modifications andequivalents, are intended by the inventors to be encompassed by theclaims. Furthermore, the discussed combination of features might not benecessary for the inventive solution.

REFERENCES

All patents, patent applications and publications referred to herein andthroughout the application are hereby incorporated by reference.

Abbott N J (2013) Blood-brain barrier structure and function and thechallenges for CNS drug delivery. J Inherit Metab Dis. 36(3):437-49.

Abulrob A, Sprong H, Van Bergen en Henegouwen P, Stanimirovic D (2005)The blood-brain barrier transmigrating single domain antibody:mechanisms of transport and antigenic epitopes in human brainendothelial cells. J Neurochem. 2005 November; 95(4):1201-14.

Arbabi-Ghahroudi, M. Desmyter A, Wyns L, Hamers R., and Muyldermans S(1997) Selection and identification of single domain antibody fragmentsfrom camel heavy-chain antibodies, FEBS Lett 414, 521-526

Arbabi-Ghahroudi, M., To, R., Gaudette, N., Hirama, T., Ding, W.,MacKenzie, R., and Tanha, J. (2009a) Protein Eng. Des. Sel. 22, 59-66.

Arbabi-Ghahroudi, M., MacKenzie, R., and Tanha, J. (2009b) Methods Mol.Biol. 525, 187-216.

Bell, A., Wang, Z. J., Arbabi-Ghahroudi, M., Chang, T. A., Durocher, Y.,Trojahn, U., Baardsnes, J., Jaramillo, M. L., Li, S., Baral, T. N.,O'Connor-McCourt, M., Mackenzie, R., and Zhang, J. (2010) Cancer Lett.289, 81-90.

Broussau, s., Jabbour, N., Lachapelle, G., Durocher, Y., Tom, R.,Transfiguracion, J., Gilbert, R. and Massie, B. (2008) Mol Ther 16,500-507.

Chothia, C., and Lesk, A. M. (1987) J. Mol. Biol. 196, 901-917.

Davies J., and L. Riechmann, Affinity improvement of single antibody VHdomains: residues in all three hypervariable regions affect antigenbinding. Immunotechnology 2 (1996) 169-179

De Kruif, J., and Logtenberg, T. (1996) J. Biol. Chem. 271, 7630-7634.

Demeule, M.; Currie, J. C.; Bertrand, Y.; Che, C.; Nguyen, T.; Regina,A.; Gabathuler, R.; Castaigne, J. P.; Beliveau, R. Involvement of thelow-density lipoprotein receptor-related protein in the transcytosis ofthe brain delivery vector angiopep-2, J. Neurochem. 2008, 106,1534-1544.

Dumoulin, M., Conrath, K., Van Meirhaighe, A., Meersman, F., Heremans,K., Frenken, L. G., Muyldermans, S., Wyns, L., and Matagne, A. (2002)Protein Sci 11, 500-515.

Durocher, Y., S. Perret, et al. (2002). “High-level and high-throughputrecombinant protein production by transient transfection ofsuspension-growing human 293-EBNA1 cells.” Nucleic Acids Res 30(2): E9.

Doyle, P. J., Arbabi-Ghahroudi, M., Gaudette, N., Furzer, G., Savard, M.E., Gleddie, S., McLean, M. D., MacKenzie, C. R., and Hall, J. C. (2008)Mol. lmmunol. 45, 3703-3713.

Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984) J. Mol.Biol. 179, 125-142

Erdlenbruch B, Alipour M, Fricker G, Miller D S, Kugler W, Eibl H,Lakomek M (2003) Alkylglycerol opening of the blood-brain barrier tosmall and large fluorescence markers in normal and C6 glioma-bearingrats and isolated rat brain capillaries. Br J Pharmacol. 140(7):1201-10.

Fenner, L., Widmer, A. F., Goy, G., Rudin, S., and Frei, R. (2008) J.Clin. Microbiol. 46, 328-330.

Gaillet, B., Gilbert, R., Broussau, S., Pilotte, A., Malenfant, F.,Mullick, A., Gamier, A., and Massie, B. (2010) Biotechnol Bioeng 106,203-215.

Gan Y, Jing Z, Stetler R A, Cao G (2013) Gene delivery with viralvectors for cerebrovascular diseases. Front Biosci (Elite Ed).5:188-203. Review.

Garberg, P.; Ball, M.; Borg, N.; Cecchelli, R.; Fenart, L.; Hurst, R.D.; Lindmark, T.; Mabondzo, A.; Nilsson, J. E.; Raub, T. J.;Stanimirovic, D.; Terasaki, T.; Oberg, J. O.; Osterberg, T. In vitromodels for the blood-brain barrier, Toxicol. In Vitro 2005, 19, 299-334.

Gergov, M.; Ojanpera, I.; Vuori, E. Simultaneous screening for 238 drugsin blood by liquid chromatography-ion spray tandem mass spectrometrywith multiple-reaction monitoring, J. Chromatogr. B Analyt. Technol.Biomed. Life Sci. 2003, 795, 41-53.

Gaillet B, Gilbert R, Amziani R, Guilbault C, Gadoury C, Caron A W,Mullick A, Gamier A, Massie B (2007) High-level recombinant proteinproduction in CHO cells using an adenoviral vector and the cumategene-switch. Biotechnol Prog. January 23(1):200-9

Gottesman et al., Ann. Rev. Biochem., 62, 385-427 (1993)

Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G.,Hamers, C., Songa, E. B., Bendahman, N., and Hamers, R. (1993) Nature363, 446-448.

Haqqani A S, Caram-Salas N, Ding W, Brunette E, Delaney C E, Baumann E,Boileau E, Stanimirovic D (2012) Multiplexed evaluation of serum and CSFpharmacokinetics of brain-targeting single-domain antibodies using aNanoLC-SRM-ILIS method. Mol Pharm. 2013 May 6; 10(5): 1542-56.

Hargreaves K M, Troullos E S, Dionne R A, Schmidt E A, Schafer S C,Joris J L (1988) Bradykinin is increased during acute and chronicinflammation: therapeutic implications. Clin Pharmacol Ther.44(6):613-21.

Huang Y L, SaIjo A, Suneson A, Hansson H A (1995) A new approach formultiple sampling of cisternal cerebrospinal fluid in rodents withminimal trauma and inflammation. J Neurosci Methods. 63(1-2):13-22.

Hussack, G., Hirama, T., Ding, W., MacKenzie, R., and Tanha, J. (2011)PLoS ONE 6, e28218.

Hussack G, Arbabi-Ghahroudi M, van Faassen H, Songer J G, Ng K K,MacKenzie R, Tanha J (2011 b) Neutralization of Clostridium difficiletoxin A with single-domain antibodies targeting the cell receptorbinding domain. J Biol Chem. 286(11): 8961-76.

Iqbal, U., Trojahn, U., Albaghdadi, H., Zhang, J., O'Connor, M.,Stanimirovic, D., Tomanek, B., Sutherland, G., and Abulrob, A. (2010)Br. J. Pharmacol. 160, 1016-1028.

Jespers, L., Schon, O., Famm, K., and Winter, G. (2004) Nat. Biotechnol.22, 1161-1165.

Kabat E A, Wu T T. Identical V region amino acid sequences and segmentsof sequences in antibodies of different specificities. Relativecontributions of VH and VL genes, minigenes, andcomplementarity-determining regions to binding of antibody-combiningsites. J Immunol. 1991; 147:1709-19.

Kim, D. Y., Kandalaft, H., Ding, W., Ryan, S., van Fassen, H., Hirama,T., Foote, S. J., MacKenzie, R., and Tanha, J. (2012) PEDS advanceaccess Aug. 30, 2012, 1-9.

Kornhuber M E, Kornhuber J, Cimniak U (1986) A method for repeated CSFsampling in the freely moving rat. J Neurosci Methods. 17(1):63-8.

Lefranc, M.-P. et al.,(2003) Dev. Comp. Immunol., 27, 55-77.

Li S, Zheng W, Kuolee R, Hirama T, Henry M, Makvandi-Nejad S, FjallmanT, Chen W, Zhang J. Pentabody-mediated antigen delivery inducesantigen-specific mucosal immune response. Mol Immunol 2009; 46:1718-26.

Lin, J. H. CSF as a surrogate for assessing CNS exposure: an industrialperspective, Curr. Drug Metab 2008, 9, 46-59.

Merritt, E. A., and Hol, W. G. (1995) Curr. Opin. Struct. Biol. 5,165-171.

Muruganandam A, Tanha J, Narang S, Stanimirovic D (2001) Selection ofphage-displayed llama single-domain antibodies that transmigrate acrosshuman blood-brain barrier endothelium. FASEB J. 2002 February;16(2):240-2.

Musher, D. M., Manhas, A., Jain, P., Nuila, F., Waqar, A., Logan, N.,Marino, B., Graviss, E. A. (2007) J. Clin. Microbiol. 45, 2737-2739.

Nhan T, Burgess A, Cho E E, Stefanovic B, Lilge L, Hynynen K. (2013)Drug delivery to the brain by focused ultrasound induced blood-brainbarrier disruption: Quantitative evaluation of enhanced permeability ofcerebral vasculature using two-photon microscopy. J Control Release.172(1):274-280.

Nicaise M, Valeio-Lepiniec M, Minard P, Desmadril M. (2004) Affinitytransfer by CDR grafting on a nonimmunoglobulin scaffold. Protein Sci.13(7): 1882-1891.

Nielsen, U. B., Adams, G. P., Weiner, L. M., and Marks, J. D. (2000)Cancer Res. 60, 6434-6440.

Nirogi, R.; Kandikere, V.; Mudigonda, K.; Bhyrapuneni, G.; Muddana, N.;Saralaya, R.; Benade, V. (2009) A simple and rapid method to collect thecerebrospinal fluid of rats and its application for the assessment ofdrug penetration into the central nervous system, J. Neurosci. Methods,178, 116-119.

Nuttall, S. D., Krishnan, U. V., Doughty, L., Pearson, K., Ryan, M. T.,Hoogenraad, N. J., Hattarki, M., Carmichael, J. A., Irving, R. A., andHudson, P. J. (2003) Eur. J. Biochem. 270, 3543-3554.

Pardridge, W. M.; Buciak, J. L.; Friden, P. M. Selective transport of ananti-transferrin receptor antibody through the blood-brain barrier invivo, J. Pharmacol. Exp. Ther. 1991, 259, 66-70.

Pardridge, W. M., Adv. Drug Delivery Reviews, 15, 5-36 (1995)

Pardridge, W. M. Drug and gene delivery to the brain: the vascularroute, Neuron. 2002, 36, 555-558.

Planche, T., Aghaizu, A., Holliman, R., Riley, P., Poloniecki, J.,Breathnach, A., and Krishna, S. (2008) Lancet Infect. Dis. 8, 777-784.

Preston E, Slinn J, Vinokourov I, Stanimirovic D. (2008) Gradedreversible opening of the rat blood-brain barrier by intracarotidinfusion of sodium caprate. J Neurosci Methods. 168(2):443-9.

Ridgway, J. B., Presta, L. G., and Carter, P. (1996) Protein Eng. 9,617-621.

Robertson C R, Flynn S P, White H S, Bulaj G (2011) Anticonvulsantneuropeptides as drug leads for neurological diseases. Nat Prod Rep.28(4):741-62.

Russmann, H., Panthel, K., Bader, R. C., Schmitt, C., and Schaumann, R.(2007) Eur. J. Clin. Microbiol. Infect. Dis. 26, 115-119.

Samani, A. A., Chevet, E., Fallavollita, L., Galipeau, J., and Brodt, P.(2004) Cancer Research 64, 3380-3385.

Samuels B. L., J. Clin. Pharmacol. Ther., 54, 421-429 (1993)

Shen, D. D.; Artru, A. A; Adkison, K. K. Principles and applicability ofCSF sampling for the assessment of CNS drug delivery andpharmacodynamics, Adv. Drug Deliv. Rev. 2004, 56, 1825-1857.

Sloan, L. M., Duresko, B. J., Gustafson, D. R., and Rosenblatt, J. E.(2008) J. Clin. Microbiol. 46, 1996-2001.

Sumbria R K, Zhou Q H, Hui E K, Lu J Z, Boado R J, Pardridge W M.(2013)Pharmacokinetics and brain uptake of an IgG-TNF decoy receptor fusionprotein following intravenous, intraperitoneal, and subcutaneousadministration in mice. Mol Pharm. 10(4):1425-31.

Tanha, J., Muruganandam, A., and Stanimirovic, D. (2003) Methods Mol.Med. 89, 435-449.

To, R., Hirama, T., Arbabi-Ghahroudi, M., MacKenzie, R., Wang, P., Xu,P., Ni, F., and Tanha, J. (2005) J. Biol. Chem. 280, 41395-41403.

Turgeon, D. K., Novicki, T.J., Quick, J., Carlson, L., Miller, P.,Ulness, B., Cent, A., Ashley, R., Larson, A., Coyle, M., Limaye, A. P.,Cookson, B. T., and Fritsche, T. R. (2003) J. Clin. Microbiol. 41,667-670.

Watanabe, T., Acta Oncol., 34, 235-241 (1995)

Xiao G, Gan L S. (2013) Receptor-mediated endocytosis and brain deliveryof therapeutic biologics. Int J Cell Biol. doi: 10.1155/2013/703545.Epub 2013 June 11.Yaksh T L, Rudy T A (1976) Chronic catheterization ofthe spinal subarachnoid space. Physiol Behay. 17(6):1031-6.

Yu, Y. J.; Zhang, Y.; Kenrick, M.; Hoyte, K.; Luk, W.; Lu, Y.; Atwal,J.; Elliott, J. M.; Prabhu, S.; Watts, R. J.; Dennis, M. S. Boostingbrain uptake of a therapeutic antibody by reducing its affinity for atranscytosis target, Sci. Transl. Med. 2011, 3, 84ra44.

Zhang, J., Li, Q., Nguyen, T.-D., Tremblay, T.-L., Stone, E., To, R.,Kelly, J., and MacKenzie, C. R. (2004a) J. Mol. Biol. 341, 161-169.

Zhang, J., Tanha, J., Hirama, T., Khiew, N. H., To, R., Tong-Sevinc, H.,Stone, E., Brisson, J. R., and MacKenzie, C. R. (2004b) J. Mol. Biol.335, 49-56.

Zhu et al., Immunology and Cell Biology (2010) 88:667-675.

European Patent No. 519596

European Patent No. 626390

U.S. Pat. No. 5,693,761

U.S. Pat. No. 5,766,886

U.S. Pat. No. 5,821,123

U.S. Pat. No. 5,859,205

U.S. Pat. No. 5,869,619

U.S. Pat. No. 6,054,297

U.S. Pat. No. 6,180,370

WO 02/057445

WO 2011/127580

WO 95/04069

WO/2004/076670

WO2003/046560

1. An isolated or purified antibody or fragment thereof specificallybinding to an Insulin-Like Growth Factor 1 Receptor (IGF1R) epitope,wherein the antibody or fragment thereof transmigrates the blood-brainbarrier, and wherein the epitope is specifically bound by the antibodyof SEQ ID NO:5.
 2. An isolated or purified antibody or fragment thereof,comprising a complementarity determining region (CDR) 1 sequence ofEYPSNFYA (SEQ ID NO:1); a CDR2 sequence of VSRDGLTT (SEQ ID NO:2); and aCDR3 sequence of AIVITGVWNKVDVNSRSYHY (SEQ ID NO:3), wherein theantibody or fragment thereof is specific for Insulin-Like Growth Factor1 Receptor (IGF1R).
 3. The isolated or purified antibody or fragmentthereof of any one of claims 1 to 2, comprising the sequenceX₁VX₂LX₃ESGGGLVQX₄GGSLRLSCX₅ASEYPSNFYAMSWX₆RQAPGKX₇X₈EX₉VX₁₀GVSRDGLTTLYADSVKGRFTX₁₁SRDNX₁₂KNTX₁₃X₁₄LQMNSX₁₅X₁₆AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX₁₇VTVSS (SEQ ID NO:4), where X₁ is E or Q; X₂ is Kor Q; X₃ iS V or E; X₄ is A or P; X₅ iS V or A; X₆ is F or V; X₇ is E orG; X₈ is R or L; X₉ is F or W; X₁₀ is A or S; X₁₁ is M or I; X₁₂ is A orS; X₁₃ is V or L; X₁₄ is D or Y; X₁₅ is V or L; X₁₆ is K or R; and X₁₇is Q or L, or a sequence substantially identical thereto.
 4. Theisolated or purified antibody or fragment thereof of any one of claims 1to 3, comprising a sequence selected from the group consisting of:(SEQ ID NO: 5) QVKLEESGGGLVQAGGSLRLSCVASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNAKNTVDLQMNSVKAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTQVTVSS; (SEQ ID NO: 6)EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 7)QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWVRQAPGKGLEWVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 8)QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKGLEFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS; (SEQ ID NO: 9)QVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVAGVSRDGLTTLYADSVKGRFTMSRDNSKNTVYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS; and (SEQ ID NO: 10)EVQLVESGGGLVQPGGSLRLSCAASEYPSNFYAMSWFRQAPGKEREFVSGVSRDGLTTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTLVTVSS.

or a sequence substantially identical thereto.
 5. The isolated orpurified antibody or fragment thereof of any one of claims 1 to 4,wherein the antibody is a single-domain antibody (sdAb).
 6. The isolatedor purified antibody or fragment thereof of claim 5, wherein the sdAb isof camelid origin.
 7. The isolated or purified antibody or fragmentthereof of any one of claims 1 to 6, wherein the antibody or fragmentthereof is in a multivalent display format.
 8. The isolated or purifiedantibody or fragment thereof of claim 7, wherein the antibody orfragment thereof is linked to a Fc fragment.
 9. The isolated or purifiedantibody or fragment thereof of claim 8, wherein the Fc fragment is themouse Fc2b or human Fc1.
 10. The isolated or purified antibody orfragment thereof of claim 9, comprising the sequence of SEQ ID NO:11,41, or
 12. 11. The isolated or purified antibody or fragment thereof ofany one of claims 2 to 10, wherein the isolated or purified antibody orfragment thereof transmigrates the blood-brain barrier.
 12. A nucleicacid molecule encoding the isolated or purified antibody or fragmentthereof of any one of claims 1 to
 11. 13. A vector comprising thenucleic acid molecule of claim
 12. 14. The isolated or purified antibodyor fragment thereof of any one of claims 1 to 13, wherein the antibodyor fragment thereof is immobilized onto a surface.
 15. The isolated orpurified antibody or fragment thereof of any one of claims 1 to 13,wherein the antibody or fragment thereof is linked to a cargo molecule.16. The isolated or purified antibody or fragment thereof of claim 15,wherein the cargo molecule has a molecular weight in the range of about1 kD to about 200 kDa.
 17. The isolated or purified antibody or fragmentthereof of claim 15 or 16, wherein the cargo molecule is a detectableagent, a therapeutic, a drug, a peptide, a growth factor, a cytokine, areceptor trap, a chemical compound, a carbohydrate moiety, an enzyme, anantibody or fragment thereof, a DNA-based molecule, a viral vector, or acytotoxic agent; one or more liposomes or nanocarriers loaded with adetectable agent, a therapeutic, a drug, a peptide, an enzyme, anantibody or fragment thereof, a DNA-based molecule, a viral vector, or acytotoxic agent; or one or more nanoparticle, nanowire, nanotube, orquantum dots.
 18. A composition comprising one or more than one isolatedor purified antibody or fragment thereof of any one of claims 1 to 11and 14 to 17 and a pharmaceutically-acceptable carrier, diluent, orexcipient.
 19. An in vitro method of detecting IGF1R, comprising a)contacting a tissue sample with one or more than one isolated orpurified antibody or fragment thereof of any one of claims 1 to 11linked to a detectable agent; and b) detecting the detectable agentlinked to the antibody or fragment thereof bound to IGF1R in the tissuesample.
 20. The method of claim 19, wherein the sample is a serumsample, a vascular tissue sample, tumour tissue sample, or a braintissue sample from a human or animal subject.
 21. The method of claim 19or 20, wherein the step of detecting (steb b) is performed using opticalimaging, immunohistochemistry, molecular diagnostic imaging, ELISA,imaging mass spectrometry, or other suitable method.
 22. An in vivomethod of detecting IGF1R expression in a subject, comprising: a)administering one or more than one isolated or purified antibody orfragment thereof of any one of claims 1 to 13 linked to a detectableagent to the subject; and b) detecting the detectable agent linked tothe antibody or fragment thereof bound to IGF1R.
 23. The method of claim22, wherein the step of detecting (steb b)) is performed using PET,SPECT, fluorescence imaging, or any other suitable method.
 24. A methodof transporting a cargo molecule across the blood-brain barrier (BBB),comprising: a) administering one or more than one isolated or purifiedantibody or fragment thereof of claim 11 linked to the cargo molecule toa subject, wherein the one or more than one antibody or fragment thereofferries the cargo molecule across the BBB.
 25. The isolated or purifiedantibody or fragment thereof of claim 24, wherein the cargo molecule hasa molecular weight in the range of about 1 kD to about 200 kDa.
 26. Themethod of claim 24 or 25, wherein the cargo molecule is a detectableagent, a therapeutic, a drug, a peptide, a growth factor, a cytokine, areceptor trap, a chemical compound, a carbohydrate moiety, an enzyme, anantibody or fragment thereof, a DNA-based molecule, a viral vector, or acytotoxic agent; one or more liposomes or nanocarriers loaded with adetectable agent, a therapeutic, a drug, a peptide, an enzyme, anantibody or fragment thereof, a DNA-based molecule, a viral vector, or acytotoxic agent; or one or more nanoparticle, nanowire, nanotube, orquantum dots.
 27. The method of any one of claims 24 to 26, wherein theadministration is intravenous (iv), subcutaneous (sc), or intramuscular(im).
 28. A method of quantifying an amount of a cargo moleculedelivered across the BBB of a subject, wherein the cargo molecule islinked to one or more than one isolated or purified antibody or fragmentthereof of claims 11, the method comprising a) collecting cerebrospinalfluid (CSF) from the subject; and b) using targeted proteomics methodsto quantify the amount of the cargo molecule linked to one or more thanone isolated or purified antibody or fragment thereof in the CSF.